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Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive... Background: Obesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood. Results: Here, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses. Conclusions: These findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression. Keywords: Obesity, Breast cancer, Lipid metabolism, Adipocytes, Metabolic crosstalk Background help cells adapt to oxidative stress and provide the Metabolic reprogramming is considered an emerging energy required for biomass synthesis, migration, and in- hallmark of cancer cells and has attracted significant vasion [2]. Much attention has centered on glucose and renewed interest both from the perspective of under- glutamine metabolism as substrates for these altered standing tumorigenesis and as a potential therapeutic pathways, in particular, as precursors for de novo lipo- target [1]. An important outcome of this metabolic shift genesis in oncogenic cell proliferation [3–5], yet the is activation of pathways that generate cellular macro- contribution of extracellular fatty acids to breast cancer molecule building blocks to support proliferation, metabolism is not well defined. including fatty acids and complex lipids for membrane The nature of tumor-stroma interactions, particularly synthesis, nucleotides for DNA/RNA synthesis, and reciprocal signaling between tumor cells and fibroblasts, amino acids for protein synthesis. These pathways also has been the subject of extensive study (see review [6]). However, more recently, this model has been broadened to consider the role of other stromal cell types (e.g., adi- * Correspondence: andrew.hoy@sydney.edu.au pocytes) and incorporate other concepts such as reciprocal Discipline of Physiology, School of Medical Sciences & Bosch Institute, The metabolic cross-talk. Martinez-Outschoorn and colleagues Hub (D17), Charles Perkins Centre, The University of Sydney, Camperdown, NSW 2006, Australia [7] have proposed a two-compartment energy model to Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Balaban et al. Cancer & Metabolism (2017) 5:1 Page 2 of 14 describe the metabolic role of tumor stroma in cancer Methods progression. In this model, tumors act as metabolic Cell culture parasites, sequestering metabolic substrates including MCF-7 (ERα positive, HTB-22, ATCC) and MDA-MB- lactate, glutamine, and fatty acids from local/stromal 231 (ERα negative, HTB-26, ATCC) human breast sources via stimulation of catabolic pathways such as cancer cells were cultured in high glucose Dulbecco’s autophagy, glycolysis, and lipolysis. This is likely to be modified Eagle’s medium (DMEM) supplemented with highly relevant in the breast where adipocytes, professional 10% fetal calf serum (FCS; HyClone, GE Healthcare Life lipid storage cells, are the predominant cell population and Sciences, USA) and 100 IU/ml penicillin and 100 IU/ml are capable of secreting significant quantities of metabolic streptomycin (Life Technologies Australia Pty Ltd., substrates such as glycerol and fatty acids. Further, there is Scoresby VIC, Australia). 3T3-L1 fibroblasts (CL-173, close juxtaposition of adipocytes and breast cancer cells ATCC) were cultured and differentiated as described pre- during early local invasion [8–10] and adipocytes are viously [22]. T47-D (HTB-113, ATCC), MDA-MB-436 proposed to be obligate partners in cancer progression [11]. (HTB-130, ATCC), MDA-MB-134 (HTB-23, ATCC), Adipocytes alter breast cancer cell growth, migration, MDA-MB-175 (HTB-25, ATCC), MDA-MB-330 (HTB- and invasion in vitro [9, 12, 13]. However, most at- 127, ATCC), MDA-MB-361 (HTB-27, ATCC), MDA-MB- tention to date has focused on the production of 468 (HTB-132, ATCC), BT-483 (HTB-121, ATCC), BT- hormones, growth factors, and cytokines by adipose 474 (HTB-20, ATCC), BT-20 (HTB-19, ATCC), and BT- tissue in tumor progression (see review [14]). 549 (HTB-122, ATCC) were cultured in RPMI (1640, Relatively, little attention has been paid to the signifi- Gibco) with 10% (v/v)FBS,1%(v/v) HEPES, and 0.25% (v/ cant potential for stromal adipocytes to provide v) human insulin. HCC-38 (CRL-2314, ATCC), HCC-70 metabolic substrates, thereby supporting breast cancer (CRL-2315, ATCC), HCC-1143 (CRL-2321, ATCC), progression. HCC-1187 (CRL-2322, ATCC), HCC-1500 (CRL-2329, Significant epidemiological evidence suggests that ATCC), and HCC1954 (CRL-2338, ATCC) were cultured obesity results in increased breast tumor size, in RPMI (1640, Gibco) with 10% (v/v)FBS,1% (v/v) increased rate of distant metastasis formation, and el- HEPES, and 1% (v/v) sodium pyruvate, 2%. MCF-10A evated mortality [15–17]. The mechanisms that (CRL-10317, ATCC) cells were cultured in HuMEC Ready underpin this relationship are yet to be defined, but medium (12752010, Invitrogen). MCF-12A (CRL-10782, in a metabolic context at least, adipocytes likely play ATCC) cells were cultured in DMEM/F12 (11320-033, an important role. However, the influence of obesity Gibco) supplemented with 5% (v/v) horse serum, EGF, in modulating the effects of adipocytes on breast hydrocortisone, cholera toxin, and bovine insulin. All cells cancer cell behavior has received limited attention. were grown at 37 °C in 5% CO . Obese adipocytes were Obesity is defined as excess accumulation of adipose generated by incubating fully differentiated adipocytes in tissue in an attempt to accommodate excess calories. basal DMEM medium supplemented with 1 mM of a Excess adiposity, in the form of increased triacylglyc- 1:2:1 palmitate (C16:0), oleate (C18:1), and linoleate erol (TAG) levels and adipocyte dysfunction, results (C18:2) (Sigma Aldrich, Castle Hill, NSW, Australia) for in increased release of fatty acids and is often associ- 24 h. Differentiated adipocytes were labeled as “lean.” All ated with hyperinsulinemia, low-grade inflammation, cell lines are validated periodically in house by and impaired adipokine secretion [18, 19]. Adipocytes Garvan Molecular Genetics using a test based on the mobilize free fatty acids from the triacylglycerol pools Powerplex 18D kit (DC1808, Promega) and tested for in a series of reactions catalyzed by adipose triglyceride mycoplasma every 3 months (MycoAlert™ mycoplasma lipase (ATGL), hormone sensitive lipase (HSL), and detection kit, Lonza). monoacylglycerol lipase (MAGL). ATGL favors TAG sub- strates and catalyzes the rate-limiting first step of lipolysis. Transwell co-culture experiments In the second step, diacylglycerol (DAG) is hydrolyzed by Co-culture experiments used a transwell system (3.0 μm HSL, which has broad substrate specificity and also hydro- pore size, Polyester (PET) Membrane; Corning Life lytic activity against TAG [20]. The orchestrated activation Sciences, Lowell, MA, USA). For experiments that of ATGL and HSL are required for complete lipolysis to assessed 3T3-L1 adipocyte biology, 5 × 10 MCF-7 or occur in adipocytes [21]. MDA-MB-231 cells were seeded in the upper chamber Here, we investigated the interaction between breast with mature adipocytes in the bottom for the indicated cancer cells and lipid-loaded “obese” adipocytes in an in times. Conversely, for experiments assessing cancer cell vitro model, focusing on the ability of breast cancer cells biology, 3T3-L1 adipocytes were grown then differenti- to mobilize stored energy-dense fatty acids from adipo- ated in the upper chamber with 5 × 10 breast cancer cytes and whether this energy transfer promotes breast cells in the bottom. Adipocytes or cancer cells cultured cancer cell proliferation and migration. alone served as controls. Balaban et al. Cancer & Metabolism (2017) 5:1 Page 3 of 14 Conditioned media generation Analytical methods Conditioned media from fully differentiated 3T3-L1 adi- Concentration of non-esterified fatty acids (NEFA-C, pocyte cells was generated by incubating cells for 24 h WAKO Diagnostics, Richmond, VA, USA) and glycerol with 10% FBS in low glucose DMEM media. 10% FBS (Free glycerol reagent, Sigma-Aldrich, Castle Hill, NSW, was substituted by 5% BSA when generating conditioned Australia) was determined using commercial kits. Adipo- media from MDA-MB-231 and MCF-7 cells. cyte triacylglycerol (TAG) content was extracted using the method of Folch et al. [26] and quantified using an Human primary mammary pre-adipocytes enzymatic colorimetric method (GPO-PAP reagent, Human breast pre-adipocytes were purchased from Roche Diagnostics). Cell protein content was determined ZenBio Inc. (North Carolina, USA) and cultured and using Pierce Micro BCA protein assay (Life Technologies differentiated in proprietary media according to the Australia Pty Ltd., Scoresby VIC, Australia). manufacturer’s instructions. Intermediary metabolism Cell proliferation and migration assays To assess co-culture intracellular substrate metabolism Lentiviral particles encoding the stable GFP expression in MCF-7 and MDA-MB-231 cells, cells were incubated vector pLV411 [23] were packaged in HEK293T cells for 4 h with low glucose DMEM medium containing 2% (CRL-3216, ATCC USA). GFP expressing MCF-7 and BSA, 1-[ C]-oleate (0.5 μCi/ml, Perkin Elmer Inc., MDA-MB-231 cells were generated by incubation with USA), 1 mM L-carnitine (Sigma), and a range of oleate pLV411 lentiviral supernatant using standard proce- (Sigma) concentrations representative of the fatty acid dures. An appropriate viral dilution was visibly selected levels observed during co-culture (0.15 mM for isolation, after serial dilution as described [24]. For proliferation 0.2 mM for lean co-culture, 0.3 mM for obese co- GFP 4 assays, MCF-7 (5 × 10 cells/well) and MDA-MB- culture groups). Fatty acid oxidation was determined by GFP 4 14 231 (5 × 10 cells/well) cells were seeded in the lower measuring CO in the culture media by the addition of chamber and the following day, cells were co-cultured an equal volume of 1 M perchloric acid and liberated with or without either lean or obese adipocytes or CO trapped in 1 N sodium hydroxide. Fatty acid in- incubated with or without either lean or obese corporation complex lipids was assessed by a Folch ex- adipocytes-conditioned media for 48 h. The percent cell traction of cellular lipids, which were concentrated confluence was continuously measured using IncuCyte- under a stream of nitrogen gas at 40 °C, resuspended in ZOOM according to the manufacturer’s instructions 100% ethanol, and transferred to scintillation vials to (Essen Bioscience, Millennium Science, Surrey Hills, measure the C activity in the organic phase. Fatty acid 14 14 NSW, Australia). uptake was calculated as the sum of CO , C in the 5 14 For cell cycle analysis, 5 × 10 MCF-7 cells were aqueous phase and C incorporation into lipid contain- cultured in either lean- or obese-conditioned media for ing organic phase of cell lysates. 24 h. After incubation, cells were fixed in cold 70% etha- For the assessment of glucose and glutamine metabol- nol at 4 °C overnight. Cells were stained with a buffer ism, the same media for oleate metabolism was used 14 14 containing propidium iodide (20 μg/ml; Sigma), and cell with the either U-[ C]-D-glucose or 1-[ C]-L-glutamine cycle analysis was assessed as previously described [25]. (0.5 μCi/ml, Perkin Elmer Inc., USA). Glucose and MDA-MB-231 cell migration was determined in a glutamine incorporated into DNA and RNA was det- scratch wound assay using the IncuCyte-ZOOM. MDA- ermined by isolating DNA and RNA using QIAGEN kits MB-231 (8 × 10 cells/well) cells were seeded and cultured according to the manufacturer’s instructions. The to 100% confluence in the lower chamber in a complete concentration of DNA/RNA was performed using a medium supplemented with 10 ng/ml mitomycin-C for 2 h NanoDrop instrument. C activity in DNA and RNA to inhibit cell proliferation. A uniform cell-free area was was achieved by adding equal volumes of DNA/RNA to created with Essen Cell Scraper (Essen Bioscience, Millen- scintillation vials. The incorporation of glucose and glu- nium Science, Surrey Hills, NSW, Australia), and the tamine into DNA and RNA was expressed as the C relative wound density (the ratio of the occupied area to activity normalized to the DNA/RNA concentration. the total area of the initial scratched region) was measured using IncuCyte during co-culture with or without either Fatty acid transfer lean or obese 3T3-L1 adipocytes. Fully differentiated adipocytes were incubated with 0.1 mM palmitate/oleate/linoleate (1:2:1) lean or 1 mM Lipid droplets visualization palmitate/oleate/linoleate (1:2:1) obese DMEM media Lean and obese 3T3-L1 adipocytes were seeded on glass supplemented with [9,10- H(N)]-oleate (0.5 μCi/ml, slides, fixed with 4% PFA, and stained for Oil Red O. Perkin Elmer Inc., USA) in 2% BSA for about 24 h. Spe- Lipid droplets were observed by using Leica DM4000. cific activity was determined by measuring cellular TAG Balaban et al. Cancer & Metabolism (2017) 5:1 Page 4 of 14 content (as above), and H in the TAG pool was assessed have largely focused on endocrine and paracrine signal- by a Folch extraction of cellular lipids followed by thin ing mechanisms (see review [33]). To determine direct layer chromatography [27]. After incubation, adipocytes functional effects of breast cancer cells on adipocyte lip- were co-cultured with either MCF-7 or MDA-MB-231 olysis, we used co-culture (Fig. 1a) and conditioned cells that were pre-seeded 1 × 10 cells/ well for 24 h. media approaches. Co-culture with MDA-MB-231 or MCF-7 and MDA-MB-231 cells were scraped in PBS and MCF-7 breast cancer cells, or exposure to conditioned H activity determined by liquid scintillation counting. media (CM) from these cell lines, increased the lipolytic rate of 3T3-L1 adipocytes, as determined by non-esteri- Western blot analysis fied fatty acid (NEFA) and glycerol release (Fig. 1b, c, re- Cell lysates were prepared as previously described [28]. spectively). Conversely, we observed an accompanying Cell lysates were subjected to SDS-PAGE, transferred to reduction in adipocyte TAG content following co-culture PVDF membranes (Merck Millipore), and then immuno- with breast cancer cells (Fig. 1d). Together, these data blotted with antibodies for anti-ATGL (#2138), anti-HSL demonstrate that breast cancer cells stimulate fatty acid (#4107), and anti-GAPDH (#2118) obtained from Cell mobilization from adipocyte TAG stores, consistent with Signaling Technology (Danvers, MA), Total OxPhos previous studies [9, 34, 35]. Complex Kit (# 458099) from Invitrogen (Life Technologies Obesity significantly influences breast cancer behavior Australia Pty Ltd), anti-14-3-3 (sc-33752) from Santa Cruz (see review [36]), and therefore, we extended these studies Biotech (Dallas, TX), and anti-CPT1A (#ab128568) from to determine whether breast cancer cell-induced fatty acid Abcam (Cambridge, MA). mobilization from adipocytes and transfer in vitro is en- hanced in the presence of obese adipocytes. To induce Gene expression survival analysis obese adipocytes, we exposed 3T3-L1 adipocytes (lean) to Analysis of CPT1A gene expression, alteration frequencies, a high-lipid environment by incubation with a physiologic- and patient outcomes (overall survival) in ER cancers (n = ally relevant fatty acid mixture for 24 h [37], a similar con- 594) from the TCGA breast cancer cohort [29] was per- cept to high-fat feeding rodents [38]. Adipocytes in this formed using the cBioPortal for Cancer Genomics [30, 31]. model displayed the cellular hallmarks of obesity, includ- ing increased lipid droplets (Fig. 1e), increased TAG con- siRNA-mediated ATGL and HSL knockdown in 3T3-L1 cells tent (Fig. 1f), and increased basal lipolysis rates (Fig. 1g). Fully differentiated 3T3-L1 adipocytes were treated with To determine whether adipocyte-derived fatty acids small interfering RNA (siRNA) as previously described accumulate in co-cultured breast cancer cells and assess [32]. Specifically, cells were electroporated with 200 nM if this is altered between cancer cells and obese adipo- scrambled (sense 5′-UUC UCC GAA CGU GUC ACG cytes, we pulsed lean and obese adipocytes with a H-la- U-3′,3′-ACG UGA CAC GUU CGG AGA A-5′) and beled fatty acid for 24 h. We then co-cultured them with ON-TARGETplus Non-Targeting Pool (Dharmacon) or breast cancer cells for a further 24 h in H-free media 200 nM pooled ON-TARGETplus Mouse Lipe siRNA– before measuring H-fatty acid transfer into breast can- SMARTpool (Dharmacon) and anti-ATGL siRNAs 1 cer cells. Adipocyte-derived H-fatty acids were taken up (sense 5′-UCA GAC GGA GAG AAC GUC AUC AUA by both MCF-7 and MDA-MB-231 cells, with MDA- U-3′,3′-AUA UGA CGU UCU CUC CGU CUG A-5′) MB-231 cells accumulating approximately twice the and 2 (5′-CCA GGC CAA UGU CUG CAG CAC AUU amount of fatty acids compared to MCF-7 cells (Fig. 1h). U-3′,3′-AAA UGU GCU GCA GAC AUU GGC CUG In both breast cancer cell lines, co-culture with obese G-5′) (Shanghai Genepharma). Cells were assayed 72 h adipocytes increased accumulation of adipocyte-derived following electroporation. H-fatty acids compared to lean adipocytes. Collectively, these data demonstrate that breast cancer cells stimulate Statistical analysis the breakdown of adipocyte TAG stores and subsequent Statistical analyses were performed with Graphpad Prism release of fatty acids, and these fatty acids are then 7.01 (Graphpad Software, San Diego, CA). Differences transferred to adjacent breast cancer cells. Importantly, among groups were assessed with appropriate statistical this effect is significantly enhanced in a cell culture tests noted in the figure legends. P ≤ 0.05 was considered model of obesity. significant. Data are reported as mean ± SEM. Adipocytes alter intermediary metabolism in breast Results cancer cells Breast cancer cells stimulate lipolysis in mature 3T3-L1 Next, we assessed the intracellular fate of fatty acids in adipocytes and accumulate adipocyte-derived fatty acids breast cancer cells co-cultured with lean and obese adi- A number of studies have described reciprocal crosstalk pocytes given the significant fatty acid transfer we ob- between cancer cells and stromal adipocytes, but these served from adipocytes to breast cancer cells (Fig. 1). Balaban et al. Cancer & Metabolism (2017) 5:1 Page 5 of 14 Fig. 1 Breast cancer cells stimulate lipolysis in mature 3T3-L1 adipocytes. a Schematic of co-culture approach. b Media non-esterified fatty acids (NEFA) concentration following co-culture of 3T3-L1 adipocytes without (isolation) or with MCF-7 and MDA-MB-231 cells for 24 h (four independent experiments performed in triplicate). c Glycerol release from 3T3-L1 adipocytes incubated in basal media, MCF-7-conditioned media (CM), and MDA- MB-231-conditioned media at the end of 24 h incubation (seven independent experiments performed in quadruplicate). d 3T3-L1 adipocytes triacylglycerol (TAG) content after 48 h co-culture with or without MCF-7 or MDA-MB-231 cells (two independent experiments performed in triplicate). e 3T3-L1 adipocyte Oil Red O staining of lipid droplets, f TAG content (three independent experiments performed in triplicate), and g NEFA release from lean (normal media) and obese (1 mM fatty acid mixture for 24 h) adipocytes compared to basal media levels (four independent experiments performed in duplicate). h Transfer of adipocyte-derived H-fatty acids from lean or obese 3T3-L1 adipocytes to MCF-7 and MDA-MB-231 cells (three independent experiments performed in duplicate). Data are presented as mean ± SEM. *P ≤ 0.05 vs. controls; #P ≤ 0.05 vs. lean. b–d and g By one-way ANOVA followed by Tukey’s multiple comparisons test, f, h by Student’s t test Following 48-h co-culture with lean 3T3-L1 adipocytes, adipocyte co-culture on glucose and glutamine both MCF-7 and MDA-MB-231 cells had increased total utilization by breast cancer cells. Uptake rates of glucose fatty acid uptake from the media and enhanced fatty acid and glutamine were not altered in MCF-7 cells following storage and mitochondrial oxidation (Fig. 2a, b). Co- adipocyte co-culture (Fig. 4a, c) but were reduced in culture with obese 3T3-L1 adipocytes had a significant MDA-MB-231 cells co-cultured with adipocytes (Fig. 4b, additional effect on this metabolic adaptation, except for d). Glucose incorporation into lipid pools was enhanced mitochondrial oxidation in MCF-7 cells (Fig. 2a, b). We in both MCF-7 and MDA-MB-231 cells co-cultured with observed induction of similar metabolic adaptations in obese 3T3-L1 adipocytes but not when co-cultured with breast cancer cells when co-cultured with differentiated lean adipocytes (Fig. 3a, b). No differences were ob- human mammary adipocytes (Fig. 2c, d). served in glutamine incorporation into lipid (Fig. 3c, d). Glucose and glutamine are key metabolic substrates Co-culturing MCF-7 or MDA-MB-231 with either lean with established roles in supporting breast cancer cell or obese 3T3-L1 adipocytes enhanced glucose oxidation growth [39]. There is significant integration of glucose in breast cancer cells by 3- and 2-fold, respectively and glutamine biochemical pathways with FA metabol- (Fig. 3a, b). Glutamine oxidation was increased in breast ism, including fatty acid synthesis from these carbon cancer cells co-cultured with obese adipocytes but not in sources. As such, we assessed the effects of lean or obese cells co-cultured with lean adipocytes (Fig. 3c, d). Balaban et al. Cancer & Metabolism (2017) 5:1 Page 6 of 14 Fig. 2 Adipocytes alter fatty acid partitioning in breast cancer cells. a MCF-7 cells and b MDA-MB-231 cells [1- C]-oleate metabolism including total uptake 14 14 14 (sum of media CO , C activity in both the aqueous and organic phases of a Folch extraction), incorporation into intracellular lipids (storage), and CO 2 2 generation (oxidation) after co-culture with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in triplicate). c MCF-7 cells and d MDA-MB-231 cells [1- C]-oleate metabolism after co-culture with or without differentiated human primary mammary pre-adipocytes for 48 h (two independent experiments performed in duplicate). Data are presented as mean ± SEM, relative to cells in isolation (dotted line). *P ≤ 0.05 compared to isolation; #P ≤ 0.05 compared to lean by one-way ANOVA followed by Tukey’s multiple comparisons test Incorporation of glucose carbons into cellular nucleotide carbons to the total lipid pool in both cell lines with glu- pools was increased post co-culture in MCF-7 cells but cose providing ~30% in MCF-7 and ~20% in MDA-MB- not in MDA-MB-231 cells (Fig. 3a, b), with no differ- 231 cells and glutamine contributing the remainder to ences observed in the incorporation of glutamine this pool (Fig. 3g). Furthermore, approximately 86% of carbons into these pools (Fig. 3c, d). These data demon- oleate taken up by MCF-7 cells is stored as a lipid, com- strate that both lean and obese 3T3-L1 adipocytes influ- pared with just 3% of glucose and 1% of glutamine with ence multiple aspects of breast cancer cell intermediary a similar pattern observed in MDA-MB-231 cells, where metabolism beyond FA metabolism. These findings the vast majority of oleate (95%) is stored as lipids, com- reinforce the importance of considering the integrated pared with 8% of glucose and 4% of glutamine. Collect- nature of metabolic biochemistry in studies of this type. ively, these data clearly demonstrate that lipid synthesis The assessment of cancer cell fatty acid metabolism is from glucose and glutamine carbons contribute only a usually limited to the generation of new fatty acids from small fraction (~35%) of the total lipid synthesis in the non-lipid sources such as glucose and glutamine (i.e., de basal state. Therefore, the reported upregulation of fatty novo lipogenesis). Using basal data from experiments acid synthesis in breast cancer [5] is not for the sole pur- presented in Figs. 2 and 3, we assessed the contribution pose of providing the bulk mass of fatty acids. of glucose, glutamine, and oleate (fatty acid) to lipid syn- thesis. We observed clear differences in basal substrate Adipocytes and fatty acids stimulate increased metabolism in the two breast cancer cell lines used in mitochondrial oxidative capacity. this study. MCF-7 cells take up glucose at a greater rate Considering the consistent increase in substrate oxida- compared with oleate and glutamine, but oleate contrib- tion by breast cancer cells following adipocyte co- utes a significantly greater amount to the cellular lipid culture, we investigated the potential role of altered pool compared to glucose and glutamine (Fig. 3e). Simi- mitochondrial function in these cells. Carnitine palmi- larly, MDA-MB-231 cells take up significantly greater toyltransferase I (CPT1) is the rate-limiting enzyme in amounts of glucose and glutamine compared to oleate mitochondrial fatty acid oxidation, mediating fatty acid with oleate contributing a much greater amount to the entry into the mitochondria [38]. As such, we assessed lipid pool compared to glucose and glutamine (Fig. 3f). CPT1 protein expression across a panel of breast cancer Upon further examination, oleate contributed ~65% of cell lines representing the main molecular sub-types and Balaban et al. Cancer & Metabolism (2017) 5:1 Page 7 of 14 Fig. 3 Altered glucose and glutamine metabolism in MDA-MB-231 and MCF-7 cells following co-culture with lean and obese adipocytes a MCF-7 14 14 14 cells and b MDA-MB-231 cells [U- C]-glucose metabolism including total uptake (sum of media CO , C activity in both the aqueous and organic phases of a Folch extraction), incorporation into intracellular lipids, CO generation (oxidation), and incorporation into DNA/RNA after co-cultured with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in duplicate, relative to cells in isolation (dotted line)). c MCF-7 cells and d MDA-MB-231 cells [1- C]-L-glutamine metabolism after co-cultured with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in duplicate, relative to cells in isolation (dotted line)). e, f Absolute rates of C-labeled substrate incorporation into intracellular lipids (lipid 14 14 synthesis) and total uptake (sum of media CO , C activity in both the aqueous and organic phases of a Folsch extraction) of various substrates in MCF-7 (e) and MDA-MB-231 (f) cells and g percent contribution of substrates to lipid synthesis in both cell lines in the basal state (three independent experiments performed in duplicate). Data are presented as mean ± SEM. a–d *P ≤ 0.05 compared to isolation; #P ≤ 0.05 compared to lean by one-way ANOVA followed by Tukey’s multiple comparisons test. e–f *P ≤ 0.05 compared to oleate; #P ≤ 0.05 compared to glucose by one-way ANOVA followed by Tukey’s multiple comparisons test observed significantly higher expression in luminal addition of oleate to growth media, further enhanced breast cancer cell lines compared to those of the basal fatty acid oxidation by MCF-7 cells but not MDA-MB- sub-type (Fig. 4a). Consistent with this expression 231 cells (Fig. 4b). This increased oxidation rate in the pattern, basal fatty acid oxidation rate is significantly presence of increased oleate availability was accompan- higher in MCF-7 cells compared to MDA-MB-231 cells ied by a marked increase in CPT1A expression in MCF- (Fig. 4b). Increasing fatty acid availability, via the 7 cells, whereas only a modest increase was observed in Balaban et al. Cancer & Metabolism (2017) 5:1 Page 8 of 14 Fig. 4 MCF-7 cells have greater CPT1 protein levels and fatty acid oxidation rates compared to MDA-MB-231 cells, and these are increased high lipid environments. a CPT1A expression in a range of normal, luminal, and basal breast cancer cell lines. b [1- C]-palmitate oxidation, c representative immunoblots of three independent experiments, and d densitometric quantitation of CPT1A protein levels in MCF-7 and MDA-MB-231 cells in the basal state and after 24 h culture in 0.3 mM oleate. e Oncoprint output showing frequency of CPT1A alteration in a TCGA cohort of ER breast cancer (n =594) (red rectangle, amplification; light red rectangle, mRNA upregulation; black rectangle, truncated mutation; green rectangle, missense mutation; gray rectangle, unaltered). f Overall survival among ER breast carcinoma TCGA cases based on CPT1A alterations. g Representative immunoblots of CPT1A and protein subunits in the mitochondrial complexes (complex II-30 kDa, complex III-Core protein 2, complex IV-subunit 1, and complex V-alpha subunit) in MCF-7 and MDA-MB-231 cells co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in duplicate). Data are presented as mean ± SEM. *P ≤ 0.05 by two-way ANOVA followed by Tukey’s multiple comparisons test MDA-MB-231 cells, which have relatively lower basal expression (P = 0.0439; Fig. 4f). By comparison, CPT1A CPT1A expression (Fig. 4c, d). Interestingly, analysis of expression was only upregulated in ~2% (2/107) basal-like 594 TCGA ER breast cancer samples showed CPT1A breast cancer samples in the TCGA dataset, and showed amplification and/or mRNA upregulation in 20% (117/ lower overall expression in the 105 unaltered cases com- + + 594) of the ER breast cancer samples (Fig. 4e). Expres- pared to ER samples (9.99 ± 0.80), similar to the expres- sion (log2) of CPT1A was significantly higher in the sion levels seen in breast cancer cell lines (Fig. 4a). altered group (11.93 ± 0.97), compared to the unaltered Next, we extended these studies to assess the molecu- −24 group (10.76 ± 0.62; P = 1.75e ). Analysis of overall lar changes in mitochondrial protein expression follow- survival in these 117 cases showed a significant decrease ing co-culture with lean and obese adipocytes. CPT1A in overall survival in patients with increased CPT1A protein levels were increased in MCF-7 cells, but not Balaban et al. Cancer & Metabolism (2017) 5:1 Page 9 of 14 MDA-MB-231, following co-culture with obese adipo- 231 cells (Fig. 5a), and this was enhanced by the pres- cytes compared with lean adipocytes (Fig. 4g). These ence of obese adipocytes (Fig. 5a). Similar effects were data suggest that the increased fatty acid oxidation observed on MDA-MB-231 cell proliferation using a capacity in breast cancer cells stimulated by adipocyte complimentary conditioned media model, as reflected by co-culture is partly determined by CPT1A protein levels, increased cell confluency determined by MTT (Fig. 2b) consistent with the established role of CPT1 as the rate- and IncuCyte (Fig. 5c). limiting step in fatty acid oxidation [38]. We also Increased distant metastasis is a common feature in observed increased expression of mitochondrial electron obese women with breast cancer [40]; as such, we transport chain complex subunits in breast cancer cells assessed the effect of lean and obese adipocytes on following adipocyte co-culture, and this effect was breast cancer cell migration. Co-culture with lean 3T3- further enhanced by co-culture with obese adipocytes L1 adipocytes increased migration of MDA-MB-231 (Fig. 4g). This observation may in part explain the cells, and this effect was significantly enhanced with observed increases in substrate oxidation in these cells obese adipocytes (Fig. 5d and Additional file 1: Figure following co-culture with adipocytes (Figs. 2 and 3). S1). Time to 50% wound closure in MDA-MB-231 cells cultured in isolation was 35 h, whereas co-culture with Adipocytes enhance breast cancer cells proliferation and lean adipocytes decreased this to 20 h and obese adipo- migration cytes reduced this further to 13 h (Fig. 5e). Hence, the Several studies have shown the role of endocrine and transfer of fatty acids from adipocytes to MDA-MB-231 paracrine signaling mechanisms in driving adipocyte- breast cancer cells in co-culture enhances both prolifera- cancer cell crosstalk [9, 12, 13]. We next sought to tion and migration of these cells. address the possibility that adipocyte-derived fatty acids Lean adipocytes increased MCF-7 proliferation during could act as a metabolic stimulus on breast cancer cell co-culture (Fig. 5f) and following exposure to adipocyte proliferation and migration in vitro. Co-culture with lean conditioned media (5G). However, we did not see any 3T3-L1 adipocytes increased proliferation of MDA-MB- additional effects of obese adipocytes on MCF-7 cell GFP Fig. 5 Adipocytes enhance breast cancer cells proliferation and migration rate. a MDA-MB-231 cell proliferation co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). b IncuCyte and c MTT assessment of MDA-MB-231 cell proliferation cultured in conditioned media from lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). d, e Migration of MDA-MB-231 cells co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed GFP in quadruplicate). f MCF-7 cell proliferation co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). g MTT assessment of MCF-7 cell proliferation cultured in conditioned media from lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). Data are presented as mean ± SEM. *P ≤ 0.05 vs. controls; #P ≤ 0.05 vs. lean a–d and f, g by two-way ANOVA repeated measure followed by Tukey’s multiple comparisons test and e by one-way ANOVA followed by Tukey’smultiple comparisons test Balaban et al. Cancer & Metabolism (2017) 5:1 Page 10 of 14 proliferation using either co-culture or conditioned and a 2-fold increase in adipocyte TAG content (Fig. 6c). media approaches (Fig. 5f, g). Together with data pre- Consequently, the transfer of adipocyte-derived fatty sented above, these observations demonstrate clear acids to MCF-7 and MDA-MB-231 cells in co-culture reciprocal interactions between breast cancer cells and was attenuated by 50 and 58% respectively following adipocytes, driving functional effects on both adipocyte siRNA-mediated knockdown of ATGL and HSL and breast cancer cell behavior. Significantly, these (Fig. 6d). effects are enhanced in the presence of obese adipocytes. Proliferation of MDA-MB-231 cells grown in condi- tioned media from ATGL/HSL knockdown adipocytes Adipocyte ATGL and HSL are required for adipocyte- was indistinguishable from cells grown in basal media mediated effects on breast cancer cell proliferation (Fig. 6e). This indicates that the adipocyte-stimulated in- Fatty acid release from adipocytes is dependent on se- crease in MDA-MB-231 cell proliferation is dependent quential hydrolysis of triacylglycerol by the lipases ATGL on ATGL/HSL mediated fatty acid release by adipocytes. and HSL [20]. We therefore determined the role of these No effect was observed on adipocyte-stimulated MCF-7 lipases in mediating adipocyte-stimulated changes in cell proliferation following ATGL/HSL knockdown breast cancer cell proliferation and migration (Fig. 5). (Fig. 6f). ATGL/HSL knockdown in adipocytes had a 3T3-L1 adipocytes were transfected with siRNAs target- small effect on adipocyte-stimulated MDA-MB-231 cell ing ATGL and HSL, which decreased protein expression migration at late time points, but this did not translate by 70 and 65%, respectively (Fig. 6a). This was accom- to differences in time to 50% wound closure (Fig. 6g, h). panied by a 70% reduction in adipocyte lipolysis (Fig. 6b) Collectively, these observations implicate an important Fig. 6 siRNA-mediated knockdown of ATGL and HSL in 3T3-L1 adipocytes. a Representative immunoblots of ATGL and HSL knockdown in 3T3-L1 adipocytes. Image is representative of six independent experiments. b NEFA secretion and c TAG content in 3T3-L1 adipocytes electroporated with either non-targeting (control) or LIPE (HSL) and PNPLA2 (ATGL) siRNAs (four independent experiments performed in triplicate). d Transfer of adipocyte-derived H-fatty acids to MCF-7 and MDA-MB-231 cells (three independent experiments performed in duplicate). e MDA-MB-231 and f MCF-7 cell proliferation co-cultured with or without ATGL/HSL knockdown 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). g, h Migration of MDA-MB-231 cells co-cultured with or without ATGL/HSL knockdown 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). Data are means and SEM. b–d *P < 0.05 compared to control siRNA with Student’s t test. e–g *P ≤ 0.05 vs. basal media, #P ≤ 0.05 compared to ATGL and HSL KD by two-way ANOVA repeated measures followed by Tukey’s multiple comparisons test and h *P ≤ 0.05 vs. basal media by one-way ANOVA followed by Tukey’s multiple comparisons test Balaban et al. Cancer & Metabolism (2017) 5:1 Page 11 of 14 role for the provisioning of adipocyte-derived fatty acids fatty acids are transferred to MCF-7 and MDA-MB-231 in supporting MDA-MB-231 cell proliferation. cells and that this rate of transfer is greater in the faster proliferating MDA-MB-231 cells compared with MCF-7 Discussion (Fig. 1). Further, this transfer is increased in a model of Significant advances in treatment strategies have sub- obesity (Fig. 1) and is mediated by adipocyte ATGL and stantially improved survival rates for many subtypes of HSL (Fig. 6). The mechanisms by which breast cancer breast cancer. However, the obesity epidemic threatens cells increase fatty acid uptake in a high lipid environ- to undermine these gains, with significant epidemio- ment are not well described. Interestingly, adipocytes logical evidence indicating obesity drives breast cancer also secrete insulin-like growth factor 1 (IGF-1) along- progression and mortality [15–17]. The metabolic mech- side fatty acids [46] and insulin-stimulated glucose and anisms that underpin this relationship between obesity fatty acid uptake is phosphoinositide 3-kinase dependent and breast cancer are yet to be identified. In this study, in skeletal muscle and adipocytes [47, 48]. Using radio- we used a combination of co-culture and conditioned metric tracing techniques, we mapped the intracellular media approaches to demonstrate significant energy fate of extracellular-derived fatty acids in breast cancer transfer from adipocytes, in the form of fatty acids, to cells in the presence of adipocytes. Adipocytes increased MCF-7 and MDA-MB-231 breast cancer cells, altering breast cancer cell fatty acid uptake, storage, and oxida- intermediary metabolism, cell proliferation, and tion (Fig. 2). Alongside the direct utilization of fatty migration. These effects were enhanced by obesity in acids by breast cancer cells, we also observed increased MDA-MB-231 cells and were largely dependent on conversion of glucose into lipid, and oxidation of glucose ATGL/HSL-mediated fatty acid release by adipocytes. and glutamine following co-culture with adipocytes It is well established that tumor cells can exert signifi- (Fig. 3). In the broader context of breast cancer cell lipid cant effects on adjacent stromal cells (see review [41]). metabolism, most attention has focused on de novo lipo- More recently, the effects of cancer cells in modifying genesis from glucose and glutamine sources via in- adipocyte biology have been described in ovarian cancer creased expression of fatty acid synthase (FASN) and [42] and prostate cancer [43]. In breast cancer, several acetyl-CoA-carboxylase 1 (ACC1) [3–5]. Here, we clearly key observations have been made showing altered adipo- show that the predominant source for lipid synthesis by cyte function. For example, adipocytes in close proximity breast cancer cells is extracellular fatty acids, not glucose to tumor in primary human samples are smaller and glutamine (Fig. 3). This demonstrates that the compared to more distal adipocytes [9, 35, 44] and tumor widely reported increase in de novo lipogenesis by breast cells induce altered adipocyte gene expression and para- cancer cells does not serve as the sole source of fatty crine signaling factor secretion [9, 35, 44, 45]. Breast can- acids for membrane synthesis and other biosynthetic re- cer cells can also significantly alter the phenotype of quirements. In fact, we show that the primary fate for mature adipocytes, including reducing TAG stores [9, 34, extracellular fatty acids is storage as complex lipids in- 35]. Building on these observations, we demonstrate that cluding glycerolipids, sphingolipids, and phospholipids, this reduced TAG store is due to a breast cancer cell-sti- consistent with a previous study showing that co-culture mulated increase in FA secretion (Fig. 1). The mechanisms of MDA-MB-231 and T47D cells with primary human that explain the effects of breast cancer cells on adipocytes omental adipocytes resulted in lipid accumulation in the are not well described but almost certainly involve se- breast cancer cells [42]. Taken together, these observa- creted factors that alter adipocyte signaling and gene ex- tions point to an important contribution by extracellular pression. For example, cancer cells stimulate expression of fatty acids, including those from local adipocytes, to the adipokines and adipocytokines (including IL-6, IL-1β, intracellular lipid pools of breast cancer cells. CCL2, CCL5, TNF-α, MCP-1, leptin), proteases, and in- Breast cancer cells exposed to adipocyte conditioned hibitors (e.g., MMP-11, PAI-1) [9, 35, 44, 45]. Further, pre- media, or in co-culture with adipocytes, have previously adipocytes in stromal vascular fraction collected from been shown to have modified proliferation, migration, mammary fat adjacent to malignant tumors had reduced and invasion [9, 12, 13, 46, 49–52]. Similar effects have differentiation capacity compared with pre-adipocytes ad- been observed in 3-D cultures [53, 54] and xenograft jacent to benign lesions [35]. models [13, 55]. A range of signaling mechanisms, in- The breast cancer cell-stimulated release of significant cluding IL-6, IGF-1, leptin, and adiponectin, have been quantities of energy dense fatty acids from adipocytes proposed to explain how mature adipocytes alter breast suggests they may represent a pool of metabolic sub- cancer cell behavior [45, 46, 56, 57], but the role of strates available to the cancer cells. This observation is adipocyte-derived fatty acids as direct metabolic sub- consistent with breast cancer cells acting as metabolic strates has not been investigated. Here, we show that parasites in the two-compartment energy model adipocyte co-culture and conditioned media stimulate described above [7]. We show that adipocyte-released MCF-7 and MDA-MB-231 cell proliferation and Balaban et al. Cancer & Metabolism (2017) 5:1 Page 12 of 14 migration (Fig. 5) and in MDA-MB-231 cells, this effect exacerbated in MDA-MB-231 cells exposed to obese adi- is dependent upon adipocyte ATGL and HSL-mediated pocytes. Taken together with the significant changes in lipolysis (Fig. 6). This was associated with increased sub- adipocyte secretory profiles in obesity, the effects of strate oxidation (Figs. 2 and 3), increased CPT1A ex- obesity on breast cancer cell behavior include a direct pression, and increased mitochondrial electron transport metabolic provisioning of substrates along with the chain subunit expression (Fig. 4). These observations well-established paracrine and endocrine signaling ef- suggest that fatty acid transfer between adipocytes and fects. Hence, our data provide an additional mechanistic cancer cells represents a significant metabolic feature of consideration in understanding the already well- the breast cancer microenvironment. Here, we demon- established link between endocrine signaling and obesity, strate for the first time a direct reciprocal metabolic and highlight the potential for targeting lipid metabolism interaction between breast cancer cells and adipocytes. in breast cancer. While increased CPT1A expression in ER breast cancer patients was associated with a significant decrease in Additional file overall survival, no data are available on obesity and Additional file 1: Figure S1. Adipocytes enhance breast cancer cells other metabolic health parameters in this patient popula- proliferation and migration rate. Representative images of IncuCyte tion. Hence, it is not possible to determine any potential analysis of migration of MDA-MB-231 cells co-cultured with or without contribution of obesity to increased CPT1A expression or “lean” or “obese” 3T3-L1 adipocytes. (TIF 14322 kb) overall survival in this cohort. To better understand the potential metabolic role of Acknowledgements Not applicable. adipocytes in mediating the effects of obesity on breast cancer cells, we generated an in vitro model of obese Funding adipocytes and used them in our co-culture and condi- AJH is supported by a Helen and Robert Ellis Postdoctoral Research Fellowship tioned media models. One of the major hallmarks of from the Sydney Medical School Foundation and funding from the University of Sydney. SB and MvG are recipients of a University of Sydney Australian obesity is expanded adipose tissue and increased fatty Postgraduate Award. MvG is supported by Cancer Institute New South Wales acid availability [58, 59]. Conditioned media generated and Sydney Catalyst. RFS is a recipient of an Australian Postgraduate Award and by adipocytes isolated from obese women (BMI 30– Baxter Family Scholarship. MS is supported by funding from the Dutch Cancer Institute KWF. JH is supported by a National Breast Cancer Foundation 35 kg/m ) have been shown to increase MCF-7 prolifer- fellowship and the University of Sydney HMR+ Implementation Fund. DNS is ation compared to lean donors [46]. Further, mammary supported by the National Health and Medical Research Council (project grant fat pad xenografts of E0771 cells had increased tumor GNT1052963), NSW Office of Science and Medical Research, Guest Family Fellowship, and Mostyn Family Foundation. volume in mice fed a high-fat diet to induce obesity and hyperinsulinemia compared with animals fed normal Availability of data and materials chow [51]. We show that co-culture of obese adipocytes Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. induces a more pronounced increase in growth and mi- gration of MDA-MB-231 cells than lean adipocytes. This Authors’ contributions was associated with increased transfer of adipocyte- SB, RFS, MvG, and MS designed and performed the experiments, analyzed the data, and edited the manuscript. LSL, HS, RC, KTC, and JH performed the derived fatty acids to MDA-MB-231 cells under obese experiments and analyzed the data. DJF, TG, and JH provided intellectual conditions, resulting in elevated TAG synthesis and input and edited the manuscript. DNS assisted in conceiving the general mitochondrial fatty acid oxidation. Increased transfer of ideas for the study, designed the experiments, analyzed the data, and wrote the manuscript. AJH conceived the general ideas for this study, designed adipocyte-derived fatty acids to MCF-7 cells was also and performed the experiments, analyzed the data, and wrote the observed under obese conditions, but this was not manuscript. All authors read and approved the final manuscript. associated with a further increase in mitochondrial fatty Competing interests acid oxidation, proliferation, or migration. These data The authors declare that they have no competing interests. highlight a potentially important role of the provision of metabolic substrates in determining the effects of adipo- Consent for publication Not applicable. cytes on breast cancer cell behavior in obesity. Ethics approval and consent to participate Conclusions Not applicable. The renewed attention to understanding the unique Author details metabolism of cancer cells has the potential to advance Discipline of Physiology, School of Medical Sciences & Bosch Institute, The clinical opportunities to exploit this tumor-specific attri- Hub (D17), Charles Perkins Centre, The University of Sydney, Camperdown, bute beyond PET imaging and into targeted therapeu- NSW 2006, Australia. Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia. Centenary Institute, The tics. In this study, we have identified a significant role University of Sydney, Camperdown, NSW 2050, Australia. Sydney Medical for fatty acids secreted from adipocytes to promote 5 School, The University of Sydney, Camperdown, NSW 2006, Australia. Faculty breast cancer cell growth and migration, which is of Medicine, University of Utrecht, Utrecht, The Netherlands. Faculty of Balaban et al. Cancer & Metabolism (2017) 5:1 Page 13 of 14 Pharmacy, The University of Sydney, Camperdown, NSW 2006, Australia. hormone-sensitive lipase are the major enzymes in adipose tissue School of Life and Environmental Sciences, Charles Perkins Centre, The triacylglycerol catabolism. J Biol Chem. 2006;281:40236–41. University of Sydney, Camperdown, NSW 2006, Australia. School of Medical 22. Zebisch K, Voigt V, Wabitsch M, Brandsch M. 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Breast Cancer Res. 2015;17:112. 58. Boden G. Free fatty acids (FFA), a link between obesity and insulin resistance. Submit your next manuscript to BioMed Central Front Biosci. 1998;3:d169–75. and we will help you at every step: 59. Wang QA, Tao C, Gupta RK, Scherer PE. Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat • We accept pre-submission inquiries Med. 2013;19:1338–44. � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer & Metabolism Springer Journals

Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration

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Springer Journals
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Copyright © 2017 by The Author(s).
Subject
Biomedicine; Cancer Research; Oncology; Metabolomics; Metabolic Diseases; Imaging / Radiology; Cell Biology
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2049-3002
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10.1186/s40170-016-0163-7
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Abstract

Background: Obesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood. Results: Here, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses. Conclusions: These findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression. Keywords: Obesity, Breast cancer, Lipid metabolism, Adipocytes, Metabolic crosstalk Background help cells adapt to oxidative stress and provide the Metabolic reprogramming is considered an emerging energy required for biomass synthesis, migration, and in- hallmark of cancer cells and has attracted significant vasion [2]. Much attention has centered on glucose and renewed interest both from the perspective of under- glutamine metabolism as substrates for these altered standing tumorigenesis and as a potential therapeutic pathways, in particular, as precursors for de novo lipo- target [1]. An important outcome of this metabolic shift genesis in oncogenic cell proliferation [3–5], yet the is activation of pathways that generate cellular macro- contribution of extracellular fatty acids to breast cancer molecule building blocks to support proliferation, metabolism is not well defined. including fatty acids and complex lipids for membrane The nature of tumor-stroma interactions, particularly synthesis, nucleotides for DNA/RNA synthesis, and reciprocal signaling between tumor cells and fibroblasts, amino acids for protein synthesis. These pathways also has been the subject of extensive study (see review [6]). However, more recently, this model has been broadened to consider the role of other stromal cell types (e.g., adi- * Correspondence: andrew.hoy@sydney.edu.au pocytes) and incorporate other concepts such as reciprocal Discipline of Physiology, School of Medical Sciences & Bosch Institute, The metabolic cross-talk. Martinez-Outschoorn and colleagues Hub (D17), Charles Perkins Centre, The University of Sydney, Camperdown, NSW 2006, Australia [7] have proposed a two-compartment energy model to Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Balaban et al. Cancer & Metabolism (2017) 5:1 Page 2 of 14 describe the metabolic role of tumor stroma in cancer Methods progression. In this model, tumors act as metabolic Cell culture parasites, sequestering metabolic substrates including MCF-7 (ERα positive, HTB-22, ATCC) and MDA-MB- lactate, glutamine, and fatty acids from local/stromal 231 (ERα negative, HTB-26, ATCC) human breast sources via stimulation of catabolic pathways such as cancer cells were cultured in high glucose Dulbecco’s autophagy, glycolysis, and lipolysis. This is likely to be modified Eagle’s medium (DMEM) supplemented with highly relevant in the breast where adipocytes, professional 10% fetal calf serum (FCS; HyClone, GE Healthcare Life lipid storage cells, are the predominant cell population and Sciences, USA) and 100 IU/ml penicillin and 100 IU/ml are capable of secreting significant quantities of metabolic streptomycin (Life Technologies Australia Pty Ltd., substrates such as glycerol and fatty acids. Further, there is Scoresby VIC, Australia). 3T3-L1 fibroblasts (CL-173, close juxtaposition of adipocytes and breast cancer cells ATCC) were cultured and differentiated as described pre- during early local invasion [8–10] and adipocytes are viously [22]. T47-D (HTB-113, ATCC), MDA-MB-436 proposed to be obligate partners in cancer progression [11]. (HTB-130, ATCC), MDA-MB-134 (HTB-23, ATCC), Adipocytes alter breast cancer cell growth, migration, MDA-MB-175 (HTB-25, ATCC), MDA-MB-330 (HTB- and invasion in vitro [9, 12, 13]. However, most at- 127, ATCC), MDA-MB-361 (HTB-27, ATCC), MDA-MB- tention to date has focused on the production of 468 (HTB-132, ATCC), BT-483 (HTB-121, ATCC), BT- hormones, growth factors, and cytokines by adipose 474 (HTB-20, ATCC), BT-20 (HTB-19, ATCC), and BT- tissue in tumor progression (see review [14]). 549 (HTB-122, ATCC) were cultured in RPMI (1640, Relatively, little attention has been paid to the signifi- Gibco) with 10% (v/v)FBS,1%(v/v) HEPES, and 0.25% (v/ cant potential for stromal adipocytes to provide v) human insulin. HCC-38 (CRL-2314, ATCC), HCC-70 metabolic substrates, thereby supporting breast cancer (CRL-2315, ATCC), HCC-1143 (CRL-2321, ATCC), progression. HCC-1187 (CRL-2322, ATCC), HCC-1500 (CRL-2329, Significant epidemiological evidence suggests that ATCC), and HCC1954 (CRL-2338, ATCC) were cultured obesity results in increased breast tumor size, in RPMI (1640, Gibco) with 10% (v/v)FBS,1% (v/v) increased rate of distant metastasis formation, and el- HEPES, and 1% (v/v) sodium pyruvate, 2%. MCF-10A evated mortality [15–17]. The mechanisms that (CRL-10317, ATCC) cells were cultured in HuMEC Ready underpin this relationship are yet to be defined, but medium (12752010, Invitrogen). MCF-12A (CRL-10782, in a metabolic context at least, adipocytes likely play ATCC) cells were cultured in DMEM/F12 (11320-033, an important role. However, the influence of obesity Gibco) supplemented with 5% (v/v) horse serum, EGF, in modulating the effects of adipocytes on breast hydrocortisone, cholera toxin, and bovine insulin. All cells cancer cell behavior has received limited attention. were grown at 37 °C in 5% CO . Obese adipocytes were Obesity is defined as excess accumulation of adipose generated by incubating fully differentiated adipocytes in tissue in an attempt to accommodate excess calories. basal DMEM medium supplemented with 1 mM of a Excess adiposity, in the form of increased triacylglyc- 1:2:1 palmitate (C16:0), oleate (C18:1), and linoleate erol (TAG) levels and adipocyte dysfunction, results (C18:2) (Sigma Aldrich, Castle Hill, NSW, Australia) for in increased release of fatty acids and is often associ- 24 h. Differentiated adipocytes were labeled as “lean.” All ated with hyperinsulinemia, low-grade inflammation, cell lines are validated periodically in house by and impaired adipokine secretion [18, 19]. Adipocytes Garvan Molecular Genetics using a test based on the mobilize free fatty acids from the triacylglycerol pools Powerplex 18D kit (DC1808, Promega) and tested for in a series of reactions catalyzed by adipose triglyceride mycoplasma every 3 months (MycoAlert™ mycoplasma lipase (ATGL), hormone sensitive lipase (HSL), and detection kit, Lonza). monoacylglycerol lipase (MAGL). ATGL favors TAG sub- strates and catalyzes the rate-limiting first step of lipolysis. Transwell co-culture experiments In the second step, diacylglycerol (DAG) is hydrolyzed by Co-culture experiments used a transwell system (3.0 μm HSL, which has broad substrate specificity and also hydro- pore size, Polyester (PET) Membrane; Corning Life lytic activity against TAG [20]. The orchestrated activation Sciences, Lowell, MA, USA). For experiments that of ATGL and HSL are required for complete lipolysis to assessed 3T3-L1 adipocyte biology, 5 × 10 MCF-7 or occur in adipocytes [21]. MDA-MB-231 cells were seeded in the upper chamber Here, we investigated the interaction between breast with mature adipocytes in the bottom for the indicated cancer cells and lipid-loaded “obese” adipocytes in an in times. Conversely, for experiments assessing cancer cell vitro model, focusing on the ability of breast cancer cells biology, 3T3-L1 adipocytes were grown then differenti- to mobilize stored energy-dense fatty acids from adipo- ated in the upper chamber with 5 × 10 breast cancer cytes and whether this energy transfer promotes breast cells in the bottom. Adipocytes or cancer cells cultured cancer cell proliferation and migration. alone served as controls. Balaban et al. Cancer & Metabolism (2017) 5:1 Page 3 of 14 Conditioned media generation Analytical methods Conditioned media from fully differentiated 3T3-L1 adi- Concentration of non-esterified fatty acids (NEFA-C, pocyte cells was generated by incubating cells for 24 h WAKO Diagnostics, Richmond, VA, USA) and glycerol with 10% FBS in low glucose DMEM media. 10% FBS (Free glycerol reagent, Sigma-Aldrich, Castle Hill, NSW, was substituted by 5% BSA when generating conditioned Australia) was determined using commercial kits. Adipo- media from MDA-MB-231 and MCF-7 cells. cyte triacylglycerol (TAG) content was extracted using the method of Folch et al. [26] and quantified using an Human primary mammary pre-adipocytes enzymatic colorimetric method (GPO-PAP reagent, Human breast pre-adipocytes were purchased from Roche Diagnostics). Cell protein content was determined ZenBio Inc. (North Carolina, USA) and cultured and using Pierce Micro BCA protein assay (Life Technologies differentiated in proprietary media according to the Australia Pty Ltd., Scoresby VIC, Australia). manufacturer’s instructions. Intermediary metabolism Cell proliferation and migration assays To assess co-culture intracellular substrate metabolism Lentiviral particles encoding the stable GFP expression in MCF-7 and MDA-MB-231 cells, cells were incubated vector pLV411 [23] were packaged in HEK293T cells for 4 h with low glucose DMEM medium containing 2% (CRL-3216, ATCC USA). GFP expressing MCF-7 and BSA, 1-[ C]-oleate (0.5 μCi/ml, Perkin Elmer Inc., MDA-MB-231 cells were generated by incubation with USA), 1 mM L-carnitine (Sigma), and a range of oleate pLV411 lentiviral supernatant using standard proce- (Sigma) concentrations representative of the fatty acid dures. An appropriate viral dilution was visibly selected levels observed during co-culture (0.15 mM for isolation, after serial dilution as described [24]. For proliferation 0.2 mM for lean co-culture, 0.3 mM for obese co- GFP 4 assays, MCF-7 (5 × 10 cells/well) and MDA-MB- culture groups). Fatty acid oxidation was determined by GFP 4 14 231 (5 × 10 cells/well) cells were seeded in the lower measuring CO in the culture media by the addition of chamber and the following day, cells were co-cultured an equal volume of 1 M perchloric acid and liberated with or without either lean or obese adipocytes or CO trapped in 1 N sodium hydroxide. Fatty acid in- incubated with or without either lean or obese corporation complex lipids was assessed by a Folch ex- adipocytes-conditioned media for 48 h. The percent cell traction of cellular lipids, which were concentrated confluence was continuously measured using IncuCyte- under a stream of nitrogen gas at 40 °C, resuspended in ZOOM according to the manufacturer’s instructions 100% ethanol, and transferred to scintillation vials to (Essen Bioscience, Millennium Science, Surrey Hills, measure the C activity in the organic phase. Fatty acid 14 14 NSW, Australia). uptake was calculated as the sum of CO , C in the 5 14 For cell cycle analysis, 5 × 10 MCF-7 cells were aqueous phase and C incorporation into lipid contain- cultured in either lean- or obese-conditioned media for ing organic phase of cell lysates. 24 h. After incubation, cells were fixed in cold 70% etha- For the assessment of glucose and glutamine metabol- nol at 4 °C overnight. Cells were stained with a buffer ism, the same media for oleate metabolism was used 14 14 containing propidium iodide (20 μg/ml; Sigma), and cell with the either U-[ C]-D-glucose or 1-[ C]-L-glutamine cycle analysis was assessed as previously described [25]. (0.5 μCi/ml, Perkin Elmer Inc., USA). Glucose and MDA-MB-231 cell migration was determined in a glutamine incorporated into DNA and RNA was det- scratch wound assay using the IncuCyte-ZOOM. MDA- ermined by isolating DNA and RNA using QIAGEN kits MB-231 (8 × 10 cells/well) cells were seeded and cultured according to the manufacturer’s instructions. The to 100% confluence in the lower chamber in a complete concentration of DNA/RNA was performed using a medium supplemented with 10 ng/ml mitomycin-C for 2 h NanoDrop instrument. C activity in DNA and RNA to inhibit cell proliferation. A uniform cell-free area was was achieved by adding equal volumes of DNA/RNA to created with Essen Cell Scraper (Essen Bioscience, Millen- scintillation vials. The incorporation of glucose and glu- nium Science, Surrey Hills, NSW, Australia), and the tamine into DNA and RNA was expressed as the C relative wound density (the ratio of the occupied area to activity normalized to the DNA/RNA concentration. the total area of the initial scratched region) was measured using IncuCyte during co-culture with or without either Fatty acid transfer lean or obese 3T3-L1 adipocytes. Fully differentiated adipocytes were incubated with 0.1 mM palmitate/oleate/linoleate (1:2:1) lean or 1 mM Lipid droplets visualization palmitate/oleate/linoleate (1:2:1) obese DMEM media Lean and obese 3T3-L1 adipocytes were seeded on glass supplemented with [9,10- H(N)]-oleate (0.5 μCi/ml, slides, fixed with 4% PFA, and stained for Oil Red O. Perkin Elmer Inc., USA) in 2% BSA for about 24 h. Spe- Lipid droplets were observed by using Leica DM4000. cific activity was determined by measuring cellular TAG Balaban et al. Cancer & Metabolism (2017) 5:1 Page 4 of 14 content (as above), and H in the TAG pool was assessed have largely focused on endocrine and paracrine signal- by a Folch extraction of cellular lipids followed by thin ing mechanisms (see review [33]). To determine direct layer chromatography [27]. After incubation, adipocytes functional effects of breast cancer cells on adipocyte lip- were co-cultured with either MCF-7 or MDA-MB-231 olysis, we used co-culture (Fig. 1a) and conditioned cells that were pre-seeded 1 × 10 cells/ well for 24 h. media approaches. Co-culture with MDA-MB-231 or MCF-7 and MDA-MB-231 cells were scraped in PBS and MCF-7 breast cancer cells, or exposure to conditioned H activity determined by liquid scintillation counting. media (CM) from these cell lines, increased the lipolytic rate of 3T3-L1 adipocytes, as determined by non-esteri- Western blot analysis fied fatty acid (NEFA) and glycerol release (Fig. 1b, c, re- Cell lysates were prepared as previously described [28]. spectively). Conversely, we observed an accompanying Cell lysates were subjected to SDS-PAGE, transferred to reduction in adipocyte TAG content following co-culture PVDF membranes (Merck Millipore), and then immuno- with breast cancer cells (Fig. 1d). Together, these data blotted with antibodies for anti-ATGL (#2138), anti-HSL demonstrate that breast cancer cells stimulate fatty acid (#4107), and anti-GAPDH (#2118) obtained from Cell mobilization from adipocyte TAG stores, consistent with Signaling Technology (Danvers, MA), Total OxPhos previous studies [9, 34, 35]. Complex Kit (# 458099) from Invitrogen (Life Technologies Obesity significantly influences breast cancer behavior Australia Pty Ltd), anti-14-3-3 (sc-33752) from Santa Cruz (see review [36]), and therefore, we extended these studies Biotech (Dallas, TX), and anti-CPT1A (#ab128568) from to determine whether breast cancer cell-induced fatty acid Abcam (Cambridge, MA). mobilization from adipocytes and transfer in vitro is en- hanced in the presence of obese adipocytes. To induce Gene expression survival analysis obese adipocytes, we exposed 3T3-L1 adipocytes (lean) to Analysis of CPT1A gene expression, alteration frequencies, a high-lipid environment by incubation with a physiologic- and patient outcomes (overall survival) in ER cancers (n = ally relevant fatty acid mixture for 24 h [37], a similar con- 594) from the TCGA breast cancer cohort [29] was per- cept to high-fat feeding rodents [38]. Adipocytes in this formed using the cBioPortal for Cancer Genomics [30, 31]. model displayed the cellular hallmarks of obesity, includ- ing increased lipid droplets (Fig. 1e), increased TAG con- siRNA-mediated ATGL and HSL knockdown in 3T3-L1 cells tent (Fig. 1f), and increased basal lipolysis rates (Fig. 1g). Fully differentiated 3T3-L1 adipocytes were treated with To determine whether adipocyte-derived fatty acids small interfering RNA (siRNA) as previously described accumulate in co-cultured breast cancer cells and assess [32]. Specifically, cells were electroporated with 200 nM if this is altered between cancer cells and obese adipo- scrambled (sense 5′-UUC UCC GAA CGU GUC ACG cytes, we pulsed lean and obese adipocytes with a H-la- U-3′,3′-ACG UGA CAC GUU CGG AGA A-5′) and beled fatty acid for 24 h. We then co-cultured them with ON-TARGETplus Non-Targeting Pool (Dharmacon) or breast cancer cells for a further 24 h in H-free media 200 nM pooled ON-TARGETplus Mouse Lipe siRNA– before measuring H-fatty acid transfer into breast can- SMARTpool (Dharmacon) and anti-ATGL siRNAs 1 cer cells. Adipocyte-derived H-fatty acids were taken up (sense 5′-UCA GAC GGA GAG AAC GUC AUC AUA by both MCF-7 and MDA-MB-231 cells, with MDA- U-3′,3′-AUA UGA CGU UCU CUC CGU CUG A-5′) MB-231 cells accumulating approximately twice the and 2 (5′-CCA GGC CAA UGU CUG CAG CAC AUU amount of fatty acids compared to MCF-7 cells (Fig. 1h). U-3′,3′-AAA UGU GCU GCA GAC AUU GGC CUG In both breast cancer cell lines, co-culture with obese G-5′) (Shanghai Genepharma). Cells were assayed 72 h adipocytes increased accumulation of adipocyte-derived following electroporation. H-fatty acids compared to lean adipocytes. Collectively, these data demonstrate that breast cancer cells stimulate Statistical analysis the breakdown of adipocyte TAG stores and subsequent Statistical analyses were performed with Graphpad Prism release of fatty acids, and these fatty acids are then 7.01 (Graphpad Software, San Diego, CA). Differences transferred to adjacent breast cancer cells. Importantly, among groups were assessed with appropriate statistical this effect is significantly enhanced in a cell culture tests noted in the figure legends. P ≤ 0.05 was considered model of obesity. significant. Data are reported as mean ± SEM. Adipocytes alter intermediary metabolism in breast Results cancer cells Breast cancer cells stimulate lipolysis in mature 3T3-L1 Next, we assessed the intracellular fate of fatty acids in adipocytes and accumulate adipocyte-derived fatty acids breast cancer cells co-cultured with lean and obese adi- A number of studies have described reciprocal crosstalk pocytes given the significant fatty acid transfer we ob- between cancer cells and stromal adipocytes, but these served from adipocytes to breast cancer cells (Fig. 1). Balaban et al. Cancer & Metabolism (2017) 5:1 Page 5 of 14 Fig. 1 Breast cancer cells stimulate lipolysis in mature 3T3-L1 adipocytes. a Schematic of co-culture approach. b Media non-esterified fatty acids (NEFA) concentration following co-culture of 3T3-L1 adipocytes without (isolation) or with MCF-7 and MDA-MB-231 cells for 24 h (four independent experiments performed in triplicate). c Glycerol release from 3T3-L1 adipocytes incubated in basal media, MCF-7-conditioned media (CM), and MDA- MB-231-conditioned media at the end of 24 h incubation (seven independent experiments performed in quadruplicate). d 3T3-L1 adipocytes triacylglycerol (TAG) content after 48 h co-culture with or without MCF-7 or MDA-MB-231 cells (two independent experiments performed in triplicate). e 3T3-L1 adipocyte Oil Red O staining of lipid droplets, f TAG content (three independent experiments performed in triplicate), and g NEFA release from lean (normal media) and obese (1 mM fatty acid mixture for 24 h) adipocytes compared to basal media levels (four independent experiments performed in duplicate). h Transfer of adipocyte-derived H-fatty acids from lean or obese 3T3-L1 adipocytes to MCF-7 and MDA-MB-231 cells (three independent experiments performed in duplicate). Data are presented as mean ± SEM. *P ≤ 0.05 vs. controls; #P ≤ 0.05 vs. lean. b–d and g By one-way ANOVA followed by Tukey’s multiple comparisons test, f, h by Student’s t test Following 48-h co-culture with lean 3T3-L1 adipocytes, adipocyte co-culture on glucose and glutamine both MCF-7 and MDA-MB-231 cells had increased total utilization by breast cancer cells. Uptake rates of glucose fatty acid uptake from the media and enhanced fatty acid and glutamine were not altered in MCF-7 cells following storage and mitochondrial oxidation (Fig. 2a, b). Co- adipocyte co-culture (Fig. 4a, c) but were reduced in culture with obese 3T3-L1 adipocytes had a significant MDA-MB-231 cells co-cultured with adipocytes (Fig. 4b, additional effect on this metabolic adaptation, except for d). Glucose incorporation into lipid pools was enhanced mitochondrial oxidation in MCF-7 cells (Fig. 2a, b). We in both MCF-7 and MDA-MB-231 cells co-cultured with observed induction of similar metabolic adaptations in obese 3T3-L1 adipocytes but not when co-cultured with breast cancer cells when co-cultured with differentiated lean adipocytes (Fig. 3a, b). No differences were ob- human mammary adipocytes (Fig. 2c, d). served in glutamine incorporation into lipid (Fig. 3c, d). Glucose and glutamine are key metabolic substrates Co-culturing MCF-7 or MDA-MB-231 with either lean with established roles in supporting breast cancer cell or obese 3T3-L1 adipocytes enhanced glucose oxidation growth [39]. There is significant integration of glucose in breast cancer cells by 3- and 2-fold, respectively and glutamine biochemical pathways with FA metabol- (Fig. 3a, b). Glutamine oxidation was increased in breast ism, including fatty acid synthesis from these carbon cancer cells co-cultured with obese adipocytes but not in sources. As such, we assessed the effects of lean or obese cells co-cultured with lean adipocytes (Fig. 3c, d). Balaban et al. Cancer & Metabolism (2017) 5:1 Page 6 of 14 Fig. 2 Adipocytes alter fatty acid partitioning in breast cancer cells. a MCF-7 cells and b MDA-MB-231 cells [1- C]-oleate metabolism including total uptake 14 14 14 (sum of media CO , C activity in both the aqueous and organic phases of a Folch extraction), incorporation into intracellular lipids (storage), and CO 2 2 generation (oxidation) after co-culture with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in triplicate). c MCF-7 cells and d MDA-MB-231 cells [1- C]-oleate metabolism after co-culture with or without differentiated human primary mammary pre-adipocytes for 48 h (two independent experiments performed in duplicate). Data are presented as mean ± SEM, relative to cells in isolation (dotted line). *P ≤ 0.05 compared to isolation; #P ≤ 0.05 compared to lean by one-way ANOVA followed by Tukey’s multiple comparisons test Incorporation of glucose carbons into cellular nucleotide carbons to the total lipid pool in both cell lines with glu- pools was increased post co-culture in MCF-7 cells but cose providing ~30% in MCF-7 and ~20% in MDA-MB- not in MDA-MB-231 cells (Fig. 3a, b), with no differ- 231 cells and glutamine contributing the remainder to ences observed in the incorporation of glutamine this pool (Fig. 3g). Furthermore, approximately 86% of carbons into these pools (Fig. 3c, d). These data demon- oleate taken up by MCF-7 cells is stored as a lipid, com- strate that both lean and obese 3T3-L1 adipocytes influ- pared with just 3% of glucose and 1% of glutamine with ence multiple aspects of breast cancer cell intermediary a similar pattern observed in MDA-MB-231 cells, where metabolism beyond FA metabolism. These findings the vast majority of oleate (95%) is stored as lipids, com- reinforce the importance of considering the integrated pared with 8% of glucose and 4% of glutamine. Collect- nature of metabolic biochemistry in studies of this type. ively, these data clearly demonstrate that lipid synthesis The assessment of cancer cell fatty acid metabolism is from glucose and glutamine carbons contribute only a usually limited to the generation of new fatty acids from small fraction (~35%) of the total lipid synthesis in the non-lipid sources such as glucose and glutamine (i.e., de basal state. Therefore, the reported upregulation of fatty novo lipogenesis). Using basal data from experiments acid synthesis in breast cancer [5] is not for the sole pur- presented in Figs. 2 and 3, we assessed the contribution pose of providing the bulk mass of fatty acids. of glucose, glutamine, and oleate (fatty acid) to lipid syn- thesis. We observed clear differences in basal substrate Adipocytes and fatty acids stimulate increased metabolism in the two breast cancer cell lines used in mitochondrial oxidative capacity. this study. MCF-7 cells take up glucose at a greater rate Considering the consistent increase in substrate oxida- compared with oleate and glutamine, but oleate contrib- tion by breast cancer cells following adipocyte co- utes a significantly greater amount to the cellular lipid culture, we investigated the potential role of altered pool compared to glucose and glutamine (Fig. 3e). Simi- mitochondrial function in these cells. Carnitine palmi- larly, MDA-MB-231 cells take up significantly greater toyltransferase I (CPT1) is the rate-limiting enzyme in amounts of glucose and glutamine compared to oleate mitochondrial fatty acid oxidation, mediating fatty acid with oleate contributing a much greater amount to the entry into the mitochondria [38]. As such, we assessed lipid pool compared to glucose and glutamine (Fig. 3f). CPT1 protein expression across a panel of breast cancer Upon further examination, oleate contributed ~65% of cell lines representing the main molecular sub-types and Balaban et al. Cancer & Metabolism (2017) 5:1 Page 7 of 14 Fig. 3 Altered glucose and glutamine metabolism in MDA-MB-231 and MCF-7 cells following co-culture with lean and obese adipocytes a MCF-7 14 14 14 cells and b MDA-MB-231 cells [U- C]-glucose metabolism including total uptake (sum of media CO , C activity in both the aqueous and organic phases of a Folch extraction), incorporation into intracellular lipids, CO generation (oxidation), and incorporation into DNA/RNA after co-cultured with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in duplicate, relative to cells in isolation (dotted line)). c MCF-7 cells and d MDA-MB-231 cells [1- C]-L-glutamine metabolism after co-cultured with or without 3T3-L1 adipocytes for 48 h (three independent experiments performed in duplicate, relative to cells in isolation (dotted line)). e, f Absolute rates of C-labeled substrate incorporation into intracellular lipids (lipid 14 14 synthesis) and total uptake (sum of media CO , C activity in both the aqueous and organic phases of a Folsch extraction) of various substrates in MCF-7 (e) and MDA-MB-231 (f) cells and g percent contribution of substrates to lipid synthesis in both cell lines in the basal state (three independent experiments performed in duplicate). Data are presented as mean ± SEM. a–d *P ≤ 0.05 compared to isolation; #P ≤ 0.05 compared to lean by one-way ANOVA followed by Tukey’s multiple comparisons test. e–f *P ≤ 0.05 compared to oleate; #P ≤ 0.05 compared to glucose by one-way ANOVA followed by Tukey’s multiple comparisons test observed significantly higher expression in luminal addition of oleate to growth media, further enhanced breast cancer cell lines compared to those of the basal fatty acid oxidation by MCF-7 cells but not MDA-MB- sub-type (Fig. 4a). Consistent with this expression 231 cells (Fig. 4b). This increased oxidation rate in the pattern, basal fatty acid oxidation rate is significantly presence of increased oleate availability was accompan- higher in MCF-7 cells compared to MDA-MB-231 cells ied by a marked increase in CPT1A expression in MCF- (Fig. 4b). Increasing fatty acid availability, via the 7 cells, whereas only a modest increase was observed in Balaban et al. Cancer & Metabolism (2017) 5:1 Page 8 of 14 Fig. 4 MCF-7 cells have greater CPT1 protein levels and fatty acid oxidation rates compared to MDA-MB-231 cells, and these are increased high lipid environments. a CPT1A expression in a range of normal, luminal, and basal breast cancer cell lines. b [1- C]-palmitate oxidation, c representative immunoblots of three independent experiments, and d densitometric quantitation of CPT1A protein levels in MCF-7 and MDA-MB-231 cells in the basal state and after 24 h culture in 0.3 mM oleate. e Oncoprint output showing frequency of CPT1A alteration in a TCGA cohort of ER breast cancer (n =594) (red rectangle, amplification; light red rectangle, mRNA upregulation; black rectangle, truncated mutation; green rectangle, missense mutation; gray rectangle, unaltered). f Overall survival among ER breast carcinoma TCGA cases based on CPT1A alterations. g Representative immunoblots of CPT1A and protein subunits in the mitochondrial complexes (complex II-30 kDa, complex III-Core protein 2, complex IV-subunit 1, and complex V-alpha subunit) in MCF-7 and MDA-MB-231 cells co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in duplicate). Data are presented as mean ± SEM. *P ≤ 0.05 by two-way ANOVA followed by Tukey’s multiple comparisons test MDA-MB-231 cells, which have relatively lower basal expression (P = 0.0439; Fig. 4f). By comparison, CPT1A CPT1A expression (Fig. 4c, d). Interestingly, analysis of expression was only upregulated in ~2% (2/107) basal-like 594 TCGA ER breast cancer samples showed CPT1A breast cancer samples in the TCGA dataset, and showed amplification and/or mRNA upregulation in 20% (117/ lower overall expression in the 105 unaltered cases com- + + 594) of the ER breast cancer samples (Fig. 4e). Expres- pared to ER samples (9.99 ± 0.80), similar to the expres- sion (log2) of CPT1A was significantly higher in the sion levels seen in breast cancer cell lines (Fig. 4a). altered group (11.93 ± 0.97), compared to the unaltered Next, we extended these studies to assess the molecu- −24 group (10.76 ± 0.62; P = 1.75e ). Analysis of overall lar changes in mitochondrial protein expression follow- survival in these 117 cases showed a significant decrease ing co-culture with lean and obese adipocytes. CPT1A in overall survival in patients with increased CPT1A protein levels were increased in MCF-7 cells, but not Balaban et al. Cancer & Metabolism (2017) 5:1 Page 9 of 14 MDA-MB-231, following co-culture with obese adipo- 231 cells (Fig. 5a), and this was enhanced by the pres- cytes compared with lean adipocytes (Fig. 4g). These ence of obese adipocytes (Fig. 5a). Similar effects were data suggest that the increased fatty acid oxidation observed on MDA-MB-231 cell proliferation using a capacity in breast cancer cells stimulated by adipocyte complimentary conditioned media model, as reflected by co-culture is partly determined by CPT1A protein levels, increased cell confluency determined by MTT (Fig. 2b) consistent with the established role of CPT1 as the rate- and IncuCyte (Fig. 5c). limiting step in fatty acid oxidation [38]. We also Increased distant metastasis is a common feature in observed increased expression of mitochondrial electron obese women with breast cancer [40]; as such, we transport chain complex subunits in breast cancer cells assessed the effect of lean and obese adipocytes on following adipocyte co-culture, and this effect was breast cancer cell migration. Co-culture with lean 3T3- further enhanced by co-culture with obese adipocytes L1 adipocytes increased migration of MDA-MB-231 (Fig. 4g). This observation may in part explain the cells, and this effect was significantly enhanced with observed increases in substrate oxidation in these cells obese adipocytes (Fig. 5d and Additional file 1: Figure following co-culture with adipocytes (Figs. 2 and 3). S1). Time to 50% wound closure in MDA-MB-231 cells cultured in isolation was 35 h, whereas co-culture with Adipocytes enhance breast cancer cells proliferation and lean adipocytes decreased this to 20 h and obese adipo- migration cytes reduced this further to 13 h (Fig. 5e). Hence, the Several studies have shown the role of endocrine and transfer of fatty acids from adipocytes to MDA-MB-231 paracrine signaling mechanisms in driving adipocyte- breast cancer cells in co-culture enhances both prolifera- cancer cell crosstalk [9, 12, 13]. We next sought to tion and migration of these cells. address the possibility that adipocyte-derived fatty acids Lean adipocytes increased MCF-7 proliferation during could act as a metabolic stimulus on breast cancer cell co-culture (Fig. 5f) and following exposure to adipocyte proliferation and migration in vitro. Co-culture with lean conditioned media (5G). However, we did not see any 3T3-L1 adipocytes increased proliferation of MDA-MB- additional effects of obese adipocytes on MCF-7 cell GFP Fig. 5 Adipocytes enhance breast cancer cells proliferation and migration rate. a MDA-MB-231 cell proliferation co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). b IncuCyte and c MTT assessment of MDA-MB-231 cell proliferation cultured in conditioned media from lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). d, e Migration of MDA-MB-231 cells co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed GFP in quadruplicate). f MCF-7 cell proliferation co-cultured with or without lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). g MTT assessment of MCF-7 cell proliferation cultured in conditioned media from lean or obese 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). Data are presented as mean ± SEM. *P ≤ 0.05 vs. controls; #P ≤ 0.05 vs. lean a–d and f, g by two-way ANOVA repeated measure followed by Tukey’s multiple comparisons test and e by one-way ANOVA followed by Tukey’smultiple comparisons test Balaban et al. Cancer & Metabolism (2017) 5:1 Page 10 of 14 proliferation using either co-culture or conditioned and a 2-fold increase in adipocyte TAG content (Fig. 6c). media approaches (Fig. 5f, g). Together with data pre- Consequently, the transfer of adipocyte-derived fatty sented above, these observations demonstrate clear acids to MCF-7 and MDA-MB-231 cells in co-culture reciprocal interactions between breast cancer cells and was attenuated by 50 and 58% respectively following adipocytes, driving functional effects on both adipocyte siRNA-mediated knockdown of ATGL and HSL and breast cancer cell behavior. Significantly, these (Fig. 6d). effects are enhanced in the presence of obese adipocytes. Proliferation of MDA-MB-231 cells grown in condi- tioned media from ATGL/HSL knockdown adipocytes Adipocyte ATGL and HSL are required for adipocyte- was indistinguishable from cells grown in basal media mediated effects on breast cancer cell proliferation (Fig. 6e). This indicates that the adipocyte-stimulated in- Fatty acid release from adipocytes is dependent on se- crease in MDA-MB-231 cell proliferation is dependent quential hydrolysis of triacylglycerol by the lipases ATGL on ATGL/HSL mediated fatty acid release by adipocytes. and HSL [20]. We therefore determined the role of these No effect was observed on adipocyte-stimulated MCF-7 lipases in mediating adipocyte-stimulated changes in cell proliferation following ATGL/HSL knockdown breast cancer cell proliferation and migration (Fig. 5). (Fig. 6f). ATGL/HSL knockdown in adipocytes had a 3T3-L1 adipocytes were transfected with siRNAs target- small effect on adipocyte-stimulated MDA-MB-231 cell ing ATGL and HSL, which decreased protein expression migration at late time points, but this did not translate by 70 and 65%, respectively (Fig. 6a). This was accom- to differences in time to 50% wound closure (Fig. 6g, h). panied by a 70% reduction in adipocyte lipolysis (Fig. 6b) Collectively, these observations implicate an important Fig. 6 siRNA-mediated knockdown of ATGL and HSL in 3T3-L1 adipocytes. a Representative immunoblots of ATGL and HSL knockdown in 3T3-L1 adipocytes. Image is representative of six independent experiments. b NEFA secretion and c TAG content in 3T3-L1 adipocytes electroporated with either non-targeting (control) or LIPE (HSL) and PNPLA2 (ATGL) siRNAs (four independent experiments performed in triplicate). d Transfer of adipocyte-derived H-fatty acids to MCF-7 and MDA-MB-231 cells (three independent experiments performed in duplicate). e MDA-MB-231 and f MCF-7 cell proliferation co-cultured with or without ATGL/HSL knockdown 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). g, h Migration of MDA-MB-231 cells co-cultured with or without ATGL/HSL knockdown 3T3-L1 adipocytes (three independent experiments performed in quadruplicate). Data are means and SEM. b–d *P < 0.05 compared to control siRNA with Student’s t test. e–g *P ≤ 0.05 vs. basal media, #P ≤ 0.05 compared to ATGL and HSL KD by two-way ANOVA repeated measures followed by Tukey’s multiple comparisons test and h *P ≤ 0.05 vs. basal media by one-way ANOVA followed by Tukey’s multiple comparisons test Balaban et al. Cancer & Metabolism (2017) 5:1 Page 11 of 14 role for the provisioning of adipocyte-derived fatty acids fatty acids are transferred to MCF-7 and MDA-MB-231 in supporting MDA-MB-231 cell proliferation. cells and that this rate of transfer is greater in the faster proliferating MDA-MB-231 cells compared with MCF-7 Discussion (Fig. 1). Further, this transfer is increased in a model of Significant advances in treatment strategies have sub- obesity (Fig. 1) and is mediated by adipocyte ATGL and stantially improved survival rates for many subtypes of HSL (Fig. 6). The mechanisms by which breast cancer breast cancer. However, the obesity epidemic threatens cells increase fatty acid uptake in a high lipid environ- to undermine these gains, with significant epidemio- ment are not well described. Interestingly, adipocytes logical evidence indicating obesity drives breast cancer also secrete insulin-like growth factor 1 (IGF-1) along- progression and mortality [15–17]. The metabolic mech- side fatty acids [46] and insulin-stimulated glucose and anisms that underpin this relationship between obesity fatty acid uptake is phosphoinositide 3-kinase dependent and breast cancer are yet to be identified. In this study, in skeletal muscle and adipocytes [47, 48]. Using radio- we used a combination of co-culture and conditioned metric tracing techniques, we mapped the intracellular media approaches to demonstrate significant energy fate of extracellular-derived fatty acids in breast cancer transfer from adipocytes, in the form of fatty acids, to cells in the presence of adipocytes. Adipocytes increased MCF-7 and MDA-MB-231 breast cancer cells, altering breast cancer cell fatty acid uptake, storage, and oxida- intermediary metabolism, cell proliferation, and tion (Fig. 2). Alongside the direct utilization of fatty migration. These effects were enhanced by obesity in acids by breast cancer cells, we also observed increased MDA-MB-231 cells and were largely dependent on conversion of glucose into lipid, and oxidation of glucose ATGL/HSL-mediated fatty acid release by adipocytes. and glutamine following co-culture with adipocytes It is well established that tumor cells can exert signifi- (Fig. 3). In the broader context of breast cancer cell lipid cant effects on adjacent stromal cells (see review [41]). metabolism, most attention has focused on de novo lipo- More recently, the effects of cancer cells in modifying genesis from glucose and glutamine sources via in- adipocyte biology have been described in ovarian cancer creased expression of fatty acid synthase (FASN) and [42] and prostate cancer [43]. In breast cancer, several acetyl-CoA-carboxylase 1 (ACC1) [3–5]. Here, we clearly key observations have been made showing altered adipo- show that the predominant source for lipid synthesis by cyte function. For example, adipocytes in close proximity breast cancer cells is extracellular fatty acids, not glucose to tumor in primary human samples are smaller and glutamine (Fig. 3). This demonstrates that the compared to more distal adipocytes [9, 35, 44] and tumor widely reported increase in de novo lipogenesis by breast cells induce altered adipocyte gene expression and para- cancer cells does not serve as the sole source of fatty crine signaling factor secretion [9, 35, 44, 45]. Breast can- acids for membrane synthesis and other biosynthetic re- cer cells can also significantly alter the phenotype of quirements. In fact, we show that the primary fate for mature adipocytes, including reducing TAG stores [9, 34, extracellular fatty acids is storage as complex lipids in- 35]. Building on these observations, we demonstrate that cluding glycerolipids, sphingolipids, and phospholipids, this reduced TAG store is due to a breast cancer cell-sti- consistent with a previous study showing that co-culture mulated increase in FA secretion (Fig. 1). The mechanisms of MDA-MB-231 and T47D cells with primary human that explain the effects of breast cancer cells on adipocytes omental adipocytes resulted in lipid accumulation in the are not well described but almost certainly involve se- breast cancer cells [42]. Taken together, these observa- creted factors that alter adipocyte signaling and gene ex- tions point to an important contribution by extracellular pression. For example, cancer cells stimulate expression of fatty acids, including those from local adipocytes, to the adipokines and adipocytokines (including IL-6, IL-1β, intracellular lipid pools of breast cancer cells. CCL2, CCL5, TNF-α, MCP-1, leptin), proteases, and in- Breast cancer cells exposed to adipocyte conditioned hibitors (e.g., MMP-11, PAI-1) [9, 35, 44, 45]. Further, pre- media, or in co-culture with adipocytes, have previously adipocytes in stromal vascular fraction collected from been shown to have modified proliferation, migration, mammary fat adjacent to malignant tumors had reduced and invasion [9, 12, 13, 46, 49–52]. Similar effects have differentiation capacity compared with pre-adipocytes ad- been observed in 3-D cultures [53, 54] and xenograft jacent to benign lesions [35]. models [13, 55]. A range of signaling mechanisms, in- The breast cancer cell-stimulated release of significant cluding IL-6, IGF-1, leptin, and adiponectin, have been quantities of energy dense fatty acids from adipocytes proposed to explain how mature adipocytes alter breast suggests they may represent a pool of metabolic sub- cancer cell behavior [45, 46, 56, 57], but the role of strates available to the cancer cells. This observation is adipocyte-derived fatty acids as direct metabolic sub- consistent with breast cancer cells acting as metabolic strates has not been investigated. Here, we show that parasites in the two-compartment energy model adipocyte co-culture and conditioned media stimulate described above [7]. We show that adipocyte-released MCF-7 and MDA-MB-231 cell proliferation and Balaban et al. Cancer & Metabolism (2017) 5:1 Page 12 of 14 migration (Fig. 5) and in MDA-MB-231 cells, this effect exacerbated in MDA-MB-231 cells exposed to obese adi- is dependent upon adipocyte ATGL and HSL-mediated pocytes. Taken together with the significant changes in lipolysis (Fig. 6). This was associated with increased sub- adipocyte secretory profiles in obesity, the effects of strate oxidation (Figs. 2 and 3), increased CPT1A ex- obesity on breast cancer cell behavior include a direct pression, and increased mitochondrial electron transport metabolic provisioning of substrates along with the chain subunit expression (Fig. 4). These observations well-established paracrine and endocrine signaling ef- suggest that fatty acid transfer between adipocytes and fects. Hence, our data provide an additional mechanistic cancer cells represents a significant metabolic feature of consideration in understanding the already well- the breast cancer microenvironment. Here, we demon- established link between endocrine signaling and obesity, strate for the first time a direct reciprocal metabolic and highlight the potential for targeting lipid metabolism interaction between breast cancer cells and adipocytes. in breast cancer. While increased CPT1A expression in ER breast cancer patients was associated with a significant decrease in Additional file overall survival, no data are available on obesity and Additional file 1: Figure S1. Adipocytes enhance breast cancer cells other metabolic health parameters in this patient popula- proliferation and migration rate. Representative images of IncuCyte tion. Hence, it is not possible to determine any potential analysis of migration of MDA-MB-231 cells co-cultured with or without contribution of obesity to increased CPT1A expression or “lean” or “obese” 3T3-L1 adipocytes. (TIF 14322 kb) overall survival in this cohort. To better understand the potential metabolic role of Acknowledgements Not applicable. adipocytes in mediating the effects of obesity on breast cancer cells, we generated an in vitro model of obese Funding adipocytes and used them in our co-culture and condi- AJH is supported by a Helen and Robert Ellis Postdoctoral Research Fellowship tioned media models. One of the major hallmarks of from the Sydney Medical School Foundation and funding from the University of Sydney. SB and MvG are recipients of a University of Sydney Australian obesity is expanded adipose tissue and increased fatty Postgraduate Award. MvG is supported by Cancer Institute New South Wales acid availability [58, 59]. Conditioned media generated and Sydney Catalyst. RFS is a recipient of an Australian Postgraduate Award and by adipocytes isolated from obese women (BMI 30– Baxter Family Scholarship. MS is supported by funding from the Dutch Cancer Institute KWF. JH is supported by a National Breast Cancer Foundation 35 kg/m ) have been shown to increase MCF-7 prolifer- fellowship and the University of Sydney HMR+ Implementation Fund. DNS is ation compared to lean donors [46]. Further, mammary supported by the National Health and Medical Research Council (project grant fat pad xenografts of E0771 cells had increased tumor GNT1052963), NSW Office of Science and Medical Research, Guest Family Fellowship, and Mostyn Family Foundation. volume in mice fed a high-fat diet to induce obesity and hyperinsulinemia compared with animals fed normal Availability of data and materials chow [51]. We show that co-culture of obese adipocytes Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. induces a more pronounced increase in growth and mi- gration of MDA-MB-231 cells than lean adipocytes. This Authors’ contributions was associated with increased transfer of adipocyte- SB, RFS, MvG, and MS designed and performed the experiments, analyzed the data, and edited the manuscript. LSL, HS, RC, KTC, and JH performed the derived fatty acids to MDA-MB-231 cells under obese experiments and analyzed the data. DJF, TG, and JH provided intellectual conditions, resulting in elevated TAG synthesis and input and edited the manuscript. DNS assisted in conceiving the general mitochondrial fatty acid oxidation. Increased transfer of ideas for the study, designed the experiments, analyzed the data, and wrote the manuscript. AJH conceived the general ideas for this study, designed adipocyte-derived fatty acids to MCF-7 cells was also and performed the experiments, analyzed the data, and wrote the observed under obese conditions, but this was not manuscript. All authors read and approved the final manuscript. associated with a further increase in mitochondrial fatty Competing interests acid oxidation, proliferation, or migration. These data The authors declare that they have no competing interests. highlight a potentially important role of the provision of metabolic substrates in determining the effects of adipo- Consent for publication Not applicable. cytes on breast cancer cell behavior in obesity. Ethics approval and consent to participate Conclusions Not applicable. The renewed attention to understanding the unique Author details metabolism of cancer cells has the potential to advance Discipline of Physiology, School of Medical Sciences & Bosch Institute, The clinical opportunities to exploit this tumor-specific attri- Hub (D17), Charles Perkins Centre, The University of Sydney, Camperdown, bute beyond PET imaging and into targeted therapeu- NSW 2006, Australia. Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia. Centenary Institute, The tics. In this study, we have identified a significant role University of Sydney, Camperdown, NSW 2050, Australia. Sydney Medical for fatty acids secreted from adipocytes to promote 5 School, The University of Sydney, Camperdown, NSW 2006, Australia. Faculty breast cancer cell growth and migration, which is of Medicine, University of Utrecht, Utrecht, The Netherlands. Faculty of Balaban et al. Cancer & Metabolism (2017) 5:1 Page 13 of 14 Pharmacy, The University of Sydney, Camperdown, NSW 2006, Australia. hormone-sensitive lipase are the major enzymes in adipose tissue School of Life and Environmental Sciences, Charles Perkins Centre, The triacylglycerol catabolism. J Biol Chem. 2006;281:40236–41. University of Sydney, Camperdown, NSW 2006, Australia. School of Medical 22. Zebisch K, Voigt V, Wabitsch M, Brandsch M. 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Breast Cancer Res. 2015;17:112. 58. Boden G. Free fatty acids (FFA), a link between obesity and insulin resistance. Submit your next manuscript to BioMed Central Front Biosci. 1998;3:d169–75. and we will help you at every step: 59. Wang QA, Tao C, Gupta RK, Scherer PE. Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat • We accept pre-submission inquiries Med. 2013;19:1338–44. � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

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