BRIEF RESEARCH REPORT published: 07 February 2019 doi: 10.3389/fphys.2019.00067 Secretory Proteome of Brown Adipocytes in Response to cAMP-Mediated Thermogenic Activation Joan Villarroya 1,2 , Rubén Cereijo 1,3 , Marta Giralt 1,3 and Francesc Villarroya 1,3* 1 Departament de Bioquímica i Biomedicina Molecular and Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain, 2 Infectious Diseases Unit, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 3 CIBER Fisiopatología de la Obesidad y Nutrición, Madrid, Spain Background: The secretory properties of brown adipose tissue are thought to contribute to the association between active brown fat and a healthy metabolic status. Although a few brown adipokines have been identified, a comprehensive knowledge of the brown adipose tissue secretome is lacking. Edited by: Paula Oliver, Methods: Here, to examine the effects of thermogenic activation of brown adipocytes on Universidad de les Illes Balears, Spain protein secretion, we used isobaric tags for relative and absolute quantification (iTRAQ) Reviewed by: Joan Ribot, analysis to determine how the secreted proteome of brown adipocytes (that detected in Universidad de les Illes Balears, Spain cell culture medium) differed in response to cAMP. Rushita Bagchi, University of Colorado Denver, Results: Our results indicated that 56 secreted proteins were up-regulated in response United States to cAMP. Of them, nearly half (29) corresponded to extracellular matrix components and *Correspondence: regulators. Several previously known adipokines, were also detected. Unexpectedly, we Francesc Villarroya fvillarroya@ub.edu also found five components of the complement system. Only 15 secreted proteins were down-regulated by cAMP; of them three were ECM-related and none was related to the Specialty section: complement system. We observed a partial concordance between the cAMP-regulated This article was submitted to release of proteins (both from proteomics and from antibody-based quantification of Integrative Physiology, a section of the journal specific proteins) and the cAMP-mediated regulation of their encoding transcript for Frontiers in Physiology the up-regulated secreted proteins. However, a stronger concordance was seen for the Received: 28 August 2018 down-regulated secreted proteins. Accepted: 21 January 2019 Published: 07 February 2019 Conclusions: The present results highlight the need to investigate previously Citation: unrecognized processes such as the role of extracellular matrix in thermogenic Villarroya J, Cereijo R, Giralt M and activation-triggered brown fat remodeling, as well as the intriguing question of how brown Villarroya F (2019) Secretory Proteome of Brown Adipocytes in Response to adipocyte-secreted complement factors contribute to the signaling properties of active cAMP-Mediated Thermogenic brown adipose tissue. Activation. Front. Physiol. 10:67. doi: 10.3389/fphys.2019.00067 Keywords: brown adipose tissue, thermogenesis, secretome, adipokine, extracellular matrix Frontiers in Physiology | www.frontiersin.org 1 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes INTRODUCTION prepared for each sample. Samples were resuspended in 500 mM triethyl ammonium bicarbonate/8M urea solution. Proteins were The high capacity of brown adipose tissue (BAT) for energy reduced with tris (2-carboxyethyl) phosphine, alkylated with expenditure and oxidation of glucose and lipids is associated with iodoacetamide and digested with porcine trypsin (Promega, protection against obesity, hyperglycemia and hyperlipidemia in Madison, WI, USA). Digested peptides were labeled with 8- rodents and possibly in humans (Bartelt et al., 2011; Giralt and plex iTRAQ reagent (AB Sciex, Wien, Austria). Pooled peptides Villarroya, 2017; Carobbio et al., 2018). However, the effects of were cleaned up in two steps: C18 clean-up (reverse phase, BAT on metabolism may also reflect the release of bioactive toptip C18) and then SCX cleanup (strong cationic exchange, factors that act in autocrine, paracrine, and even endocrine P200 toptip, PolySULFOETHYL A) (PolyLC, Columbia, MD, manners (Villarroya et al., 2017). The secretory activity of USA). Peptides were analyzed by liquid chromatography coupled white fat has long been recognized and a large number of to mass spectrometry using nanoAcquity (Waters, Milford, “white” adipokines have been identified. In contrast, the secretory MA, USA) coupled to LTQ-Orbitrap Velos mass spectrometer properties of BAT have received much less research attention. (ThermoScientific, Foster City, CA, USA). Up to the 10 most Most brown adipokines were identified from the high-level abundant peptides were selected from each MS scan and expression of genes encoding putative secreted proteins, such then fragmented using Higher Energy Collision Dissociation. as fibroblast growth factor-21 (FGF21) or interleukin-6 (IL-6) Database search was performed with Mascot search engine in active BAT (Burýsek and Houstek, 1997; Hondares et al., against SwissProt/Uniprot database. IsobariQ software (Arntzen 2011). In some cases, brown adipokine candidates were identified et al., 2011) was used to perform the relative quantitation of by screening transcriptomic data from BAT using bioinformatic proteins by iTRAQ. We used the annotation “extracellular” in tools that can predict the “secretability” of encoded factors (Wang the Gene Onthology database, TargetP 1.1 (Emanuelsson et al., et al., 2014; Verdeguer et al., 2015; Cereijo et al., 2018). Other 2007), and Secretome P 2.0 (Bendtsen et al., 2004) software to approaches have used indirect methodologies, such as signal identify secreted proteins. Crude data from proteomics analysis sequence trap (Hansen et al., 2014). However, we still lack a are available at www.adipoplast.org/datebase. comprehensive knowledge of the BAT secretome. Here, we use an updated iTRAQ-based proteomic analysis to provide the first Quantitative Real Time PCR report of a direct assessment of proteins secreted by brown RNA was extracted (Nucleospin, Macherey-Nagel, Düren, adipocytes in response to cAMP, which is the main intracellular Germany) and retrotranscribed using TaqMan Reverse mediator of BAT thermogenic activity. Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Transcript levels were determined by quantitative MATERIALS AND METHODS real-time PCR on a 7500 Real-Time PCR System (Applied Biosystems), using TaqMan assays or SyberGreen assays Cell Culture with specifically designed primers (PerlPrimer software) (see Murine C57BAT brown pre-adipocytes were supplied by Klein Supplementary Material). The mRNA levels of each gene et al. (1999) and were obtained using protocols approved of interest was normalized to 18S rRNA levels using the by the Institutional Animal Care and Use Committee, Joslin 2−1Ct method. Diabetes Center (Boston, USA). Pre-adipocytes were cultured in DMEM (Life Technologies, Grand Island, NY, USA), 10% FBS, Specific Quantification of Secreted 1% penicillin-streptomycin, for 2–3 days. Before differentiation Proteins was induced, the plates were rinsed with PBS to remove Individual proteins were quantified in brown adipocyte culture the FBS; further culture was performed in FBS-free medium. medium using specific ELISA kits (see Supplemental Table 2 for Differentiation was induced by culturing cells overnight in suppliers), except retinol-binding protein-4 (RBP4), which was DMEM plus T3 (1 nM), insulin (20 nM), IBMX (500 µM), quantified by immunoblotting, as previously described (Rosell dexamethasone (2 mM), and indomethacin (125 µM), and then et al., 2012). replacing this medium with DMEM plus T3 and insulin until brown adipocytes had fully differentiated (5–6 days). Differentiated brown adipocytes were left untreated or treated RESULTS with 1 mM dibutyryl-cAMP for 24 h. Three independent samples were used for each group. Medium was collected and cells were Differentiation and Effects of cAMP in scrapped and collected for RNA analysis. Unless otherwise noted, Brown Adipocytes Cultured in Serum-Free reagents were from Merck Sigma-Aldrich, S. Louis, MO, USA. Medium Our brown adipocyte culture protocol, adapted to allow a Comparative Proteomic Analysis iTRAQ proteomics-based analysis of the culture medium (see section Identification of proteins with differences in abundance between Materials and Methods), resulted in more than 90% adipogenic cAMP-treated and non-cAMP-treated brown adipocytes was differentiation, as assessed by the percentage of cells exhibiting performed using the isobaric tag for relative and absolute lipid droplet accumulations (Figure 1A), and expression of quantitation (iTRAQ) technology (see Supplementary Material the brown adipocyte marker gene Ucp1. Cell culture media for detailed methodology). Three independent replicates were were collected from cAMP-treated and non-treated controls Frontiers in Physiology | www.frontiersin.org 2 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes for proteomic analysis. Transcript levels of Ucp1 and Fgf21, and results were more concordant with transcript changes which encodes for a thermogenically-regulated secreted protein (Figure 1F). (Hondares et al., 2011), were strongly induced in cAMP-treated brown adipocytes (Figure 1B). This validates the experimental model used to perform the proteomic analysis. DISCUSSION To our knowledge, this report describes the first direct assessment Identification of cAMP-Regulated Secreted of the protein secretome of cultured brown adipocytes stimulated Proteins with cAMP, which triggers thermogenic activation. Thirteen A total of 71 extracellular proteins were found to differ of the proteins we detected with our iTRAQ-based method in their abundance in cAMP-treated vs. non-treated cultures coincided with those identified by Hansen et al. (2014) (Table 1). Fifty-six secreted proteins were induced by cAMP using indirect signal sequence trap method to identify brown (Figure 1C, left). Of them, 40% (22 proteins) were components adipocyte-secreted proteins in response to norepinephrine of the extracellular matrix (ECM) and 13% (7 proteins) were (Table 1). They corresponded mainly to ECM-related factors (6) matricellular proteins (non-structural proteins that are present and the miscellaneous group of proteins (5). Moreover, a recent in the ECM and play regulatory roles). Among the remaining iTRAQ-based detection of proteins secreted early after induction up-regulated secreted proteins, seven were extracellular enzymes, of 3T3-L1 white adipocyte differentiation (Ojima et al., 2016) six were adipokines, and four corresponded to components of using isobutyl-methylxanthine (a phosphodiesterase inhibitor the complement system. Finally, we observed up-regulation of 10 that raises intracellular cAMP) yielded results that were similar to varied proteins, including cytokines, transporters, and proteins of our data. Matches were seen for 41% of the ECM-related proteins unknown function. Notably, fewer secreted proteins were down- (12 of 29), 66% of the adipokines (4 of 6), and 60% of the “others” regulated by cAMP (Figure 1C, right). Of these 15 proteins, five (6 of 10) (Table 1). This similarity between the two data sets may were extracellular enzymes, three were ECM-related, one was an reflect the activation of common cAMP-dependent pathways in adipokine, and six formed a heterogeneous group of proteins adipogenic cells. with multiple or unknown functions. A remarkable finding of our study is the strong representation To ascertain whether secreted proteins underwent (around 50%) of ECM-related components in the cAMP- transcriptional regulation in response to cAMP, we measured induced secretome of brown adipocytes. A functional and the transcript levels of selected components of the distinct flexible ECM is essential for healthy expansion of WAT groups of differentially secreted proteins. We found that secreted (Sun et al., 2013). The thermogenic activation of BAT proteins that were up-regulated by cAMP did not show a causes a tissue recruitment that involves hypertrophic and systematic relationship with transcript expression (Figure 1D). hyperplastic processes (Cannon and Nedergaard, 2004). Among the ECM-related genes, the three tested collagen gene The cAMP-induced secretion of ECM components, such as transcripts (Col1a1, Col4a1, and Col4a2) failed to show cAMP- specific types of collagen and matricellular proteins, may induced up-regulation; indeed Col1a1 was down-regulated. In be relevant to the remodeling of ECM under conditions contrast, the transcript level of Lamb1 was increased by cAMP, of BAT expansion in response to thermogenic activation. consistently with the up-regulation of secreted laminin-β1 Further research will be needed to explore this novel notion in protein in cAMP-treated cultures. With respect to extracellular BAT biology. enzymes, the transcript levels of Xdh and Ppia were increased in We identified several components of the complement response to cAMP, as observed for the corresponding secreted system in the cAMP-induced secretome of brown adipocytes. proteins. The analyzed adipokine-encoding gene transcripts Hansen et al. (2014) also identified several components of the varied: Fabp4 was up-regulated, Adipoq was down-regulated, complement system in their brown adipocyte secretome analysis. and Rbp4 was unchanged. Varied changes were also observed Interestingly, a transcriptomic analysis identified complement for the transcripts encoding some of the other cAMP-induced factor-4 as one of the top secreted protein-encoding transcripts secreted proteins: cAMP increased the transcript level of Grn up-regulated in human BAT vs. WAT (Svensson et al., 2011). (granulins), but decreased that of Orm1 (orosomucoid). Among Complement factor-D (adipsin), one of the first white adipokines the transcripts encoding for secreted proteins whose abundance identified (Cook et al., 1987), was here found to be up-regulated was reduced by cAMP treatment, we found a more consistent in the secretome of cAMP-treated brown adipocytes. Further pattern: the transcript levels of Efemp1, Lpl, Lbp, and B2m research is warranted to ascertain the role of complement system were all significantly down-regulated in response to cAMP components in the signaling that originates in brown adipocytes (Figure 1E). upon thermogenic activation. Given the discordance between proteome-based detection of Our study identified several brown adipokines previously secreted proteins and the corresponding transcripts in some reported to be regulated in response to thermogenic activation cases, levels of selected proteins from distinct functional groups in adipose tissues, such as adiponectin (Hui et al., 2015), RBP4 were individually quantified. COL1A1, LAMB1, RBP4, and GRN (Rosell et al., 2012), angiotensinogen (Cassis, 1993), fatty acid proteins were confirmed to be up-regulated and LBP to be binding protein-4 (Shu et al., 2017), and lipopolysaccharide down-regulated by cAMP. However, adiponectin (ADIPOQ) binding protein (LBP) (Gavaldà-Navarro et al., 2016). These and ORM1 were not confirmed to be up-regulated by cAMP, findings further validate our proteomic analysis. However, our Frontiers in Physiology | www.frontiersin.org 3 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes FIGURE 1 | Differentiation, transcript levels, and secreted proteins in cultured brown adipocytes in response to cAMP. (A) Representative optical microscopic images of pre-adipocytes (left), and brown adipocytes left untreated or treated with cAMP for 24 h (right). (B) Relative transcript levels of Ucp1 and Fgf21 in untreated (none) and cAMP-treated (cAMP 24 h) differentiated brown adipocytes (n = 6). (C) Secreted proteins found to be up-regulated (left) and down-regulated (right) in response to cAMP were classified into six groups according to their function: ECM component, matricellular, extracellular enzymes, adipokines, complement, and others (cytokines, transport, etc). (D) Transcript levels corresponding to selected secreted proteins up-regulated by cAMP treatment of brown adipocytes. (E) Transcript levels corresponding to selected secreted proteins down-regulated by cAMP treatment of brown adipocytes. (F) Levels of secreted proteins in brown adipocyte culture medium individually quantified using specific antibody-based methods. Bars represent means ± s.e.m of six samples per group. Two-tailed unpaired Student’s t-test was used to compare means (*p < 0.05, **p < 0.01, ***p < 0.001, cAMP vs. no treatment). Frontiers in Physiology | www.frontiersin.org 4 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes TABLE 1 | List of secreted proteins found to be up-regulated (A) or down-regulated (B) upon cAMP treatment of brown adipocytes, categorized by their functions. Function UniprotKB Gene symbol Description Length cAMP-induced fold-change (A) UP REGULATED PROTEINS IN RESPONSE To cAMP ECM component P07356 Anxa2 Annexin A2 339 1.12 Q62059-3 Vcan Versican core protein 2,393 1.56 P97927 Lama4 Laminin subunit alpha-4 1,816 1.36 P02469 Lamb1 Laminin subunit beta-1# 1,786 1.51 P02468 Lamc1 Laminin subunit gamma-1 1,607 1.40 P08122 Col4a2 Collagen alpha-2(IV) chain# 1,707 1.36 Q02788 Col6a2 Collagen alpha-2(VI) chain# 1,034 1.33 P10493 Nid1 Nidogen-1# 1,245 1.35 P82198 Tgfbi Transforming growth factor-beta-induced protein 693 1.29 ig-h3 Q61554 Fbn1 Fibrillin-1# 2,871 1.28 Q3U962 Col5a2 Collagen alpha-2(V) chain# 1,497 1.26 P02463 Col4a1 Collagen alpha-1(IV) chain# 1,669 1.25 P11276 Fn1 Fibronectin 2,477 1.25 P11087 Col1a1 Collagen alpha-1(I) chain# 1,453 1.22 O35206 Col15a1 Collagen alpha-1(XV) chain 1,367 1.19 Q05793 Hspg2 Basement membrane-specific heparan sulfate 3,707 1.18 proteoglycan core protein (perlecan)*# P33434 Mmp2 72 kDa type IV collagenase 662 1.18 Q04857 Col6a1 Collagen alpha-1(VI) chain# 1,025 1.17 P08121 Col3a1 Collagen alpha-1(III) chain*# 1,464 1.12 O08746-2 Matn2 Matrilin-2 956 1.07 Q61398 Pcolce Procollagen C-endopeptidase enhancer 1*# 468 1.07 O88207 Col5a1 Collagen alpha-1(V) chain# 1,838 1.02 ECM matricellular Q08879 Fbln1 Fibulin-1 705 1.19 P37889-2 Fbln2 Fibulin-2 # 1,221 1.20 Q62009-3 Postn Periostin 838 1.20 P70663 Sparcl1 SPARC-like protein 1 650 1.14 P07214 Sparc SPARC* 302 1.10 P16110 Lgals3 Galectin-3 264 1.11 Q07797 Lgals3bp Galectin-3-binding protein 577 1.17 Extracellular enzymes Q8BND5-2 Qsox1 Sulfhydryl oxidase 1 748 1.56 P10605 Ctsb Cathepsin B# 339 1.37 P00687 Amy1 Alpha-amylase 1 511 1.04 P00688 Amy2 Pancreatic alpha-amylase 508 1.13 P21460 Cst3 Cystatin-C# 140 1.10 Q00519 Xdh Xanthine dehydrogenase/oxidase 1,335 1.07 P17742 Ppia Peptidyl-prolyl cis-trans isomerase A# 164 1.04 Adipokines Q60994 Adipoq Adiponectin# 247 1.31 Q00724 Rbp4 Retinol-binding protein 4 201 1.31 P11859 Agt Angiotensinogen# 477 1.13 P04117 Fabp4 Fatty acid-binding protein, adipocyte 132 1.02 P03953-2 Cfd Complement factor D (adipsin)# 259 1.01 Q9DD06 Rarres2 Retinoic acid receptor responder protein 2 162 1.01 (Chemerin)*# Complement P01029 C4b Complement C4-B 1,738 1.09 P04186 Cfb Complement factor B 761 1.06 P48759 Ptx3 Pentraxin-related protein PTX3 381 1.06 P01027 C3 Complement C3# 1,663 1.05 (Continued) Frontiers in Physiology | www.frontiersin.org 5 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes TABLE 1 | Continued. Function UniprotKB Gene symbol Description Length cAMP-induced fold-change Others (cytokines, P21956-2 Mfge8 Lactadherin 463 1.60 transport,...) Q62356 Fstl1 Follistatin-related protein 1*# 306 1.34 P28798 Grn Granulins 589 1.24 P17515 Cxcl10 C-X-C motif chemokine 10 98 1.20 O88968 Tcn2 Transcobalamin-2 430 1.14 Q9Z0J0 Npc2 Epididymal secretory protein E1 (Niemann-Pick)*# 149 1.09 P47879 Igfbp4 Insulin-like growth factor-binding protein 4*# 254 1.09 O88393 Tgfbr3 Transforming growth factor beta receptor type 3 850 1.08 (beta-glycan)# Q60590 Orm1 Alpha-1-acid glycoprotein 1 (orosomucoid)# 207 1.06 P97298 Serpinf1 Pigment epithelium-derived factor (Serpin1)# 417 1.05 (B) DOWN-REGULATED PROTEINS IN RESPONSE TO cAMP ECM component P28653 Bgn Biglycan*# 369 1.03 Matricellular Q8BPB5 Efemp1 EGF-containing fibulin-like extracellular matrix 493 1.35 Extracellular enzymes protein 1* P16045 Lgals1 Galectin-1 135 1.13 Q06890 Clu Clusterin 448 1.01 P10639 Txn Thioredoxin 105 1.07 P06745 Gpi Glucose-6-phosphate isomerase (neuroleukin) 558 1.06 P11152 Lpl Lipoprotein lipase* 474 1.06 P18242 Ctsd Cathepsin D 410 1.04 Adipokines Q61805 Lbp Lipopolysaccharide-binding protein 481 1.05 Others (cytokines, Q61646 Hp Haptoglobin* 347 1.15 transport,...) P13020 Gsn Gelsolin# 780 1.07 Q61207 Psap Prosaposin 557 1.07 P01887 B2m Beta-2-microglobulin# 119 1.07 P08226 Apoe Apolipoprotein E* 311 1.05 P34884 Mif Macrophage migration inhibitory factor# 115 1.01 The UniprotKB code, gene symbol, description, length (amino acids), and cAMP-induced fold-change for each detected protein are shown. *Indicates concordance with Hansen (2014), # Indicates concordance with Ojima et al. (2016). secretome data did not include other known brown adipokines, confirmed most, but not all, changes detected in the such as IL-6, FGF21, or neuregulin-4 (NRG4). This is likely to proteomics assays, even in cases when transcript levels reflect that such proteins are present at relatively low levels in were discordant. This suggests that post-transcriptional the cell culture medium (1–10 ng/ml) (Burýsek and Houstek, mechanisms are involved in the cAMP-mediated regulation of 1997; Hondares et al., 2011; Rosell et al., 2014). In contrast, protein secretion by brown adipocytes and that transcriptomic the levels of secreted proteins such as adiponectin or LBP approaches may therefore be limited in their ability to predict exceed 100 ng/ml in the culture media of adipocyte culture comprehensively the BAT secretome. Moreover, our data systems (Díaz-Delfín et al., 2012; Moreno-Navarrete et al., 2013). highlight the importance of specific validation of proteomics- The relatively low-level release of some secreted proteins by based findings using individual quantification of secreted protein brown adipocytes may be a limitation in the ability to identify candidates. secretome components using proteomic analysis of culture In conclusion, the current study opens new venues for media. research of the secretome of brown adipocytes. Beyond helping We found only partial concordance between the cAMP- direct further research on novel brown adipokine candidates, our regulated secreted proteins according to proteomics and work suggests two new research foci, namely: (a) understanding the corresponding transcript levels. The discordance was the importance of the synthesis of ECM components by particularly relevant for cAMP-induced ECM components brown adipocytes for BAT remodeling during thermogenic and adipokines, whereas cAMP-repressed secreted factors adaptation, and (b) determining the roles of the complement- showed more concordance. Moreover, individual quantification related proteins by brown adipocytes upon thermogenic of a set of secreted protein candidates responsive to cAMP activation. Frontiers in Physiology | www.frontiersin.org 6 February 2019 | Volume 10 | Article 67 Villarroya et al. Secretome of Brown Adipocytes AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS JV performed the brown adipocyte cell cultures, The proteomics work was done at the Plataforma de Proteòmica proteomic analytics, qRT-PCR, immunoblot, and del Parc Científic de Barcelona, a member of ProteoRed, PRB3 ELISA assays. RC performed the bioinformatic and is supported by grant PT17/0019/0016, funded by Instituto analysis of secretability. MG and FV designed the de Salud Carlos III (Spain) and European Region Development study and compiled the data. FV and JV wrote the Fund. Thanks are given to Dr. E. de Oliveira for support in the manuscript. proteomics analysis, and to Dr. J. Klein (University of Luebeck, Germany), and Dr. A. Whittle (University of Cambridge, UK) for FUNDING the supply of C57 brown pre-adipocytes. This work was supported by grants from the Ministerio SUPPLEMENTARY MATERIAL de Economia y Competitividad (MINECO), Spain (SAF2017- 85722R and PI17/00420), co-financed by the European Regional The Supplementary Material for this article can be found Development Fund (ERDF). JV is a Juan de la Cierva online at: https://www.frontiersin.org/articles/10.3389/fphys. post-doctoral researcher by MINECO, Spain. 2019.00067/full#supplementary-material REFERENCES Hansen, I. R. (2014). The Secretome of Brown Adipose Tissue. Doctoral thesis, Stockholm University, Stockholm. Arntzen, M. Ø., Koehler, C. J., Barsnes, H., Berven, F. S., Treumann, A., and Thiede, Hansen, I. R., Jansson, K. M., Cannon, B., and Nedergaard, J. (2014). Contrasting B. (2011). IsobariQ: software for isobaric quantitative proteomics using IPTL, effects of cold acclimation versus obesogenic diets on chemerin gene expression iTRAQ, and TMT. J. Proteome Res. 4, 913–920. doi: 10.1021/pr1009977 in brown and brite adipose tissues. 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Brown adipose YY1 deficiency activates expression of secreted conducted in the absence of any commercial or financial relationships that could proteins linked to energy expenditure and prevents diet-induced obesity. Mol. be construed as a potential conflict of interest. Cell. Biol. 36, 184–196. doi: 10.1128/MCB.00722-15 Villarroya, F., Cereijo, R., Villarroya, J., and Giralt, M. (2017). Brown adipose tissue Copyright © 2019 Villarroya, Cereijo, Giralt and Villarroya. This is an open-access as a secretory organ. Nat. Rev. Endocrinol. 13, 26–35. doi: 10.1038/nrendo.20 article distributed under the terms of the Creative Commons Attribution License (CC 16.136 BY). The use, distribution or reproduction in other forums is permitted, provided Wang, G. X., Zhao, X. Y., Meng, Z. X., Kern, M., Dietrich, A., Chen, the original author(s) and the copyright owner(s) are credited and that the original Z., et al. (2014). The brown fat-enriched secreted factor Nrg4 preserves publication in this journal is cited, in accordance with accepted academic practice. metabolic homeostasis through attenuation of hepatic lipogenesis. Nat. Med. No use, distribution or reproduction is permitted which does not comply with these 20, 1436–1443. doi: 10.1038/nm.3713 terms. Frontiers in Physiology | www.frontiersin.org 8 February 2019 | Volume 10 | Article 67