Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation

In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT “whitening ” is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity. In mammals, white adipose tissue (WAT) stores and releases lipids, whereas brown adipose tissue (BAT) oxidizes lipids to fuel thermogenesis. In obese individuals, WAT undergoes profound changes; it expands, becomes dysfunctional, and develops a low-grade inflammatory state. Importantly, BAT content and activity decline in obese subjects, mainly as a result of the conversion of brown adipocytes to white-like unilocular cells. Here, we show that BAT “whitening ” is induced by multiple factors, including high ambient temperature, leptin receptor deficiency, β-adrenergic signaling impairment, and lipase deficiency, each of which is capable of inducing macrophage infiltration, brown adipocyte death, and crown-like structure (CLS) formation. Brown-to-white conversion and increased CLS formation were most marked in BAT from adipose triglyceride lipase (Atgl)-deficient mice, where, according to transmission electron microscopy, whitened brown adipocytes contained enlarged endoplasmic reticulum, cholesterol crystals, and some degenerating mitochondria, and were surrounded by an increased number of collagen fibrils. Gene expression analysis showed that BAT whitening in Atgl-deficient mice was associated to a strong inflammatory response and NLRP3 inflammasome activation. Altogether, the present findings suggest that converted enlarged brown adipocytes are highly prone to death, which, by promoting inflammation in whitened BAT, may contribute to the typical inflammatory state seen in obesity. Obesity is a major and growing public health problem. Reports based on the BMI (weight/height2), an indirect marker of adiposity, have shown that over the past decades, obesity has been increasing worldwide (1.NCD Trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19.2 million participants.Lancet. 2016; 387: 1377-1396Abstract Full Text Full Text PDF PubMed Scopus (3108) Google Scholar). Obesity is a severe clinical problem, because it is associated with an increased risk of developing a variety of medical conditions, such as insulin resistance, hypertension, dyslipidemia, nonalcoholic fatty liver, cardiovascular disease, and even some cancers. Importantly, it is the major risk factor for type 2 diabetes (2.Klöting N. Fasshauer M. Dietrich A. Kovacs P. Schön M.R. Kern M. Stumvoll M. Blüher M. Insulin-sensitive obesity.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E506-E515Crossref PubMed Scopus (493) Google Scholar, 3.Klöting N. Fasshauer M. Dietrich A. Kovacs P. Schön M.R. Kern M. Stumvoll M. Blüher M. Insulin-sensitive obesity.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E506-E515Crossref PubMed Scopus (590) Google Scholar). Adipose tissue dysfunction and inflammation are hallmarks of morbid obesity. In obese mice and humans, adipose tissue is infiltrated by inflammatory cells and produces inflammatory mediators that link fat accumulation to cardiovascular and metabolic complications, such as insulin resistance and type 2 diabetes (4.Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7435) Google Scholar, 5.Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. et al.Chronic inflammation in fat plays a crucial role in the development of obesity related insulin resistance.J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5172) Google Scholar, 6.Cinti S. Mitchell G. Barbatelli G. Murano I. Ceresi E. Faloia E. Wang S. Fortier M. Greenberg A.S. Obin M.S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.J. 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Here, monocyte-derived macrophages positive for MAC-2 often localize around dead adipocytes and form the so-called crown-like structures (CLSs) (6.Cinti S. Mitchell G. Barbatelli G. Murano I. Ceresi E. Faloia E. Wang S. Fortier M. Greenberg A.S. Obin M.S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.J. Lipid Res. 2005; 46: 2347-2355Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar), where aggregates of activated macrophages, sometimes fused into syncytia (multinucleated giant cells), surround and clear dead adipocytes. Importantly, macrophage infiltration of obese fat positively correlates with adipocyte size and CLS density (4.Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante A.W. Obesity is associated with macrophage accumulation in adipose tissue.J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7435) Google Scholar, 5.Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. et al.Chronic inflammation in fat plays a crucial role in the development of obesity related insulin resistance.J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5172) Google Scholar, 6.Cinti S. Mitchell G. Barbatelli G. Murano I. Ceresi E. Faloia E. Wang S. Fortier M. Greenberg A.S. Obin M.S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.J. Lipid Res. 2005; 46: 2347-2355Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar, 7.Murano I. Barbatelli G. Parisani V. Latini C. Muzzonigro G. Castellucci M. Cinti S. Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.J. Lipid Res. 2008; 49: 1562-1568Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). In particular, the number of CLSs in obese fat is in direct proportion to the degree of adipocyte hypertrophy in both visceral and subcutaneous adipose tissue (7.Murano I. Barbatelli G. Parisani V. Latini C. Muzzonigro G. Castellucci M. Cinti S. Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.J. Lipid Res. 2008; 49: 1562-1568Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). However, their density is higher in visceral adipose tissue, where adipocytes are smaller, than in subcutaneous fat, where adipocytes are larger. This suggests that lipid overload results in adipocyte growth up to a critical size; growth beyond this size induces profound cell stress and leads to death. Thus, the critical size is smaller in visceral than in subcutaneous fat depots (6.Cinti S. Mitchell G. Barbatelli G. Murano I. Ceresi E. Faloia E. Wang S. Fortier M. Greenberg A.S. Obin M.S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.J. Lipid Res. 2005; 46: 2347-2355Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar, 7.Murano I. Barbatelli G. Parisani V. Latini C. Muzzonigro G. Castellucci M. Cinti S. Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.J. Lipid Res. 2008; 49: 1562-1568Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Hypertrophic adipocytes from obese mice are prone to developing organelle dysfunction, such as endoplasmic reticulum (ER) stress (10.Gregor M.F. Hotamisligil G.S. Adipocyte stress: the endo­plasmic reticulum and metabolic disease.J. Lipid Res. 2007; 48: 1905-1914Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar), mitochondrial dysfunction (11.Kusminski C.M. Scherer P.E. Mitochondrial dysfunction in white adipose tissue.Trends Endocrinol. Metab. 2012; 23: 435-443Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), and activation of the NLRP3 inflammasome pathway, suggesting that hypertrophic stressed adipocytes undergo pyroptotic cell death and initiate an inflammatory response (12.Giordano A. Murano I. Mondini E. Perugini J. Smorlesi A. Severi I. Barazzoni R. Scherer P.E. Cinti S. Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis.J. Lipid Res. 2013; 54: 2423-2436Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Notably, organelle dysfunction and inflammasome activation are more marked in visceral than in subcutaneous adipocytes (12.Giordano A. Murano I. Mondini E. Perugini J. Smorlesi A. Severi I. Barazzoni R. Scherer P.E. Cinti S. Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis.J. Lipid Res. 2013; 54: 2423-2436Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The mammalian adipose organ contains white and brown adipocytes (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar). White adipocytes, which are found in white adipose tissue (WAT), store dietary energy as triglycerides in a single large lipid droplet (unilocular adipocytes); at times of high calorie requirements, triglycerides undergo hydrolyzation and the resulting fatty acids are secreted into the bloodstream and supplied to other tissues. Brown adipocytes are the main parenchymal cell type found in brown adipose tissue (BAT). They are characterized by multiple small triglyceride droplets (multilocular adipocytes) and contain numerous large mitochondria, where uncoupling protein 1 (UCP1) enables use of the energy derived from fatty acid oxidation for heat generation (nonshivering thermogenesis) (14.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar). In normally fed mice maintained at normal ambient temperature, white and brown adipocytes are found together in several subcutaneous and visceral depots (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar, 14.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar, 15.Murano I. Barbatelli G. Giordano A. Cinti S. Noradrenergic parenchymal nerve fiber branching after cold acclimatisation correlates with brown adipocyte density in mouse adipose organ.J. Anat. 2009; 214: 171-178Crossref PubMed Scopus (154) Google Scholar, 16.Vitali A. Murano I. Zingaretti M.C. Frontini A. Ricquier D. Cinti S. The adipose organ of obesity-prone C57BL/6J mice is composed of mixed white and brown adipocytes.J. Lipid Res. 2012; 53: 619-629Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In contrast, in the adipose organ of obese animals and humans, typical brown adipocytes are barely detectable because most of them undergo a still poorly elucidated conversion to a “white-like” phenotype (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar, 17.Sbarbati A. Morroni M. Zancanaro C. Cinti S. Rat interscapular brown adipose tissue at different ages: a morphometric study.Int. J. Obes. 1991; 15: 581-587PubMed Google Scholar, 18.Cinti S. Frederich R.C. Zingaretti M.C. De Matteis R. Flier J.S. Lowell B.B. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue.Endocrinology. 1997; 138: 797-804Crossref PubMed Scopus (171) Google Scholar). Given the limited available data on the fate and possible proinflammatory role of white-like adipocytes derived from brown-to-white conversion in obese mice, the present study was performed to investigate the implications of brown-to-white-like conversion (in terms of the tendency of adipocytes to develop inflammation and to die) in the adipose depots of mice challenged with environmental, dietary, and genetic stimuli. Adipose triglyceride lipase (Atgl)-knockout (ko) mice generated by targeted homologous recombination (19.Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar) were backcrossed at least seven times to a C57BL/6J genetic background. All experiments were carried out in male 8- to10-week-old Atgl-ko mice and corresponding wild-type littermates. The adipose-specific Atgl-ko (AAKO) mice (20.Schoiswohl G. Stefanovic-Racic M. Menke M.N. Wills R.C. Surlow B.A. Basantani M.K. Sitnick M.T. Cai L. Yazbeck C.F. Stolz D.B. et al.Impact of reduced ATGL-mediated adipocyte lipolysis on obesity-associated insulin resistance and inflammation in male mice.Endocrinology. 2015; 156: 3610-3624Crossref PubMed Scopus (110) Google Scholar) are described in the supplemental Materials and Methods. Db/+ and db/db female mice aged 5 weeks were purchased from Charles River (Lecco, Italy) and used for experimental procedures at 14 weeks of age (five animals per strain). C57BL/6J female mice (Harlan, Udine, Italy) aged 12 weeks were kept at 28°C (n = 5, warm-acclimated mice) or at 6°C (n = 5, cold-acclimated mice) for 10 days to reduce and respectively increase the noradrenergic inputs to the adipose organ. Adult mice lacking β-adrenergic receptors (β-less mice) were kindly provided by Dr. B. B. Lowell (Harvard Medical School, Boston, MA) (21.Bachman E.S. Dhillon H. Zhang C.Y. Cinti S. Bianco C.A. Kobilka K.B. Lowell B.B. betaAR signaling required for diet-induced thermogenesis and obesity resistance.Science. 2002; 297: 843-845Crossref PubMed Scopus (632) Google Scholar). Animals were individually caged and maintained on a 12:12 h light/dark cycle with free access to standard pellet food and water. The animal experiments performed at the Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy were in accordance with institutional and national guidelines and were approved by the institutional Animal Ethics Board of Università Politecnica delle Marche. Experiments carried out at the Institute of Molecular Biosciences, University of Graz, Graz, Austria were approved and performed according to the guidelines of the ethics committee of the University of Graz and the Austrian Federal Ministry for Science and Research. For morphological studies, mice were euthanized with an overdose of anesthetic (Avertin; Fluka Chemie, Buchs, Switzerland) and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 5 min. BAT and WAT depots were dissected using a Zeiss OPI1 surgical microscope (Carl Zeiss, Oberkochen, Germany) and further fixed by immersion in 4% paraformaldehyde in PB overnight at 4°C. After a thorough rinse in PB, small fragments were collected for transmission electron microscopy (TEM; see below). The remaining tissue was dehydrated in ethanol, cleared in xylene, and embedded in paraffin. For molecular biology assays, animals were anesthetized with ISOflo®/isoflurane (Abbott, Abbott Park, IL) and euthanized by cervical dislocation. BAT and WAT specimens were rapidly removed, snap-frozen in liquid nitrogen, and stored at −80°C until use. Serial paraffin sections (3 μm thick) were obtained from each adipose depot, placed on glass slides, and dried. Alternate sections were used for hematoxylin and eosin staining to assess morphology, and for immunohistochemical procedures to evaluate tissue protein expression. Adipocyte size was defined as mean adipocyte area (in square micrometers) using a drawing tablet and a morphometric program (Nikon LUCIA IMAGE, Laboratory Imaging, version 4.61; Praha, Czech Republic). Tissue sections were examined with a Nikon Eclipse E800 light microscope, and digital images were captured at 20× with a Nikon DXM 1200 camera (Nikon Instruments S.p.A, Calenzano, Italy). CLS density was determined by counting the total number of (MAC-2-positive) CLSs in each section compared with the total number of adipocytes and was expressed as CLS number/10,000 adipocytes. The CLS index was calculated by dividing CLS density by mean adipocyte size. For immunohistochemistry, 3 μm thick paraffin-embedded sections of the fat depots were dewaxed; they were then reacted with 0.3% H2O2 (in methanol; 30 min) to block endogenous peroxidase, rinsed with PBS, and incubated in 3% normal serum blocking solution (in PBS; 30 min). Sections were then incubated overnight at 4°C with rat monoclonal anti-MAC-2 antibody (dilution 1:1,500; Cedarlane Laboratories, Burlington, Ontario, Canada), a marker of activated macrophages, or rabbit polyclonal anti-perilipin antibody (dilution 1:300; kindly provided by Dr. A. S. Greenberg, Boston, MA). After a thorough rinse in PBS, sections were incubated in 1:200 v/v horse anti-rat (MAC-2 schedule) or goat anti-rabbit (perilipin schedule) IgG biotinylated HRP-conjugated secondary antibody solution (Vector Laboratories, Burlingame, CA) in PBS for 30 min. Histochemical reactions were performed using a Vectastain ABC kit (Vector Laboratories) and Sigma Fast 3,3′-diaminobenzidine (Sigma-Aldrich, Vienna, Austria) as the substrate. Sections were counterstained with hematoxylin, dehydrated in ethanol, and mounted in Eukitt® mounting medium (Sigma-Aldrich). Staining was never observed when the primary antibody was omitted. The procedures applied for immunofluorescence and confocal microscopy analysis are described in the supplemental Materials and Methods. Small fragments of BAT and WAT from perfused mice were fixed in 2% glutaraldheyde-2% paraformaldehyde in PB for 4 h at room temperature, postfixed in 1% osmium tetroxide, dehydrated in a graded series of acetone, and embedded in an Epon-Araldite mixture. To determine the region of interest, semi-thin sections were cut and stained with toluidine blue. Thin sections were obtained with an MT-X Ultratome (RMC, Tucson, AZ), stained with lead citrate, and examined with a CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands). Homogenization of frozen BAT and WAT for RNA isolation was performed using an Ultra-Turrax apparatus (IKA, Staufen, Germany) and TRIzol® reagent (Life Technologies, Invitrogen, Vienna, Austria) according to standard protocols. For gene expression analyses, RNA samples were treated with DNase I (Life Technologies, Invitrogen) and reverse transcribed into single-stranded cDNA using a high-capacity reverse transcription kit (Life Technologies, Applied Biosystems, Vienna, Austria). cDNA samples were amplified using Maxima SYBR Green/ROX Master Mix 2x (Fermentas Life Science, St. Leon-Roth, Germany) and primer pairs specific for the target genes (Life Technologies, Invitrogen). The primer sequences are available on request. Real-time PCR was performed on a C1000 thermocycler using the CFX96 real-time system (Bio-Rad Laboratories GmbH, Vienna, Austria). Relative target gene expression was normalized to the ribosomal gene, 36b4, and calculated using the ΔΔCt method (22.Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123424) Google Scholar). The procedures regarding RNA isolation and quantitative (q)PCR of AAKO mice are described in the supplemental Materials and Methods. Total RNA from BAT was reverse transcribed with a high-capacity reverse transcription kit (Life Technologies, Applied Biosystems) using the reverse Xbp-1 specific primer, reverse 5′-GAGGCAACAGTGTCAGAGTCC-3′, to amplify Xbp-1 cDNA. For Xbp-1 cDNA amplification, conventional PCR was performed using gene-specific primers for Xbp-1 (forward 5′-GAACCAGGAGTTAAGAACACG-3′ and reverse 5′-GAGGCAACAGTGTCA­GAGTCC-3′). PCR reactions were run using Phusion DNA polymerase (Biozym, Vienna, Austria). Primers were designed to amplify both unspliced and unconventionally spliced Xbp-1 in the same reaction. Samples were separated on 3% agarose gel. DNA gels were documented using Image Quant 300 (GE Healthcare Europe GmbH, Vienna, Austria). Whole genomic DNA and mitochondrial (mt)DNA were extracted from adipose tissue depots using DNeasy blood and tissue kit (Qiagen Vertriebs GmbH, Vienna, Austria) according to the manufacturer's instructions. mtDNA copy number was determined with quantitative real-time PCR using SYBR green and specific primers for the mitochondrial-encoded cytochrome c oxidase 1 (Mt-Co1) and the single copy nuclear gene, NADH dehydrogenase (ubiquinone) flavoprotein 1 (Ndufv1), as control, as described previously (23.Amthor H. Macharia R. Navarrete R. Schuelke M. Brown S.C. Otto A. Voit T. Muntoni F. Vrbóva G. Partridge T. et al.Lack of myostatin results in excessive muscle growth but impaired force generation.Proc. Natl. Acad. Sci. USA. 2007; 104: 1835-1840Crossref PubMed Scopus (301) Google Scholar). DNA samples were amplified using Maxima SYBR Green/ROX Master Mix 2x (Fermentas Life Science) and gene-specific primer pairs (Life Technologies, Invitrogen). PCR reactions were run on an ABI One Step PLUS system (Life Technologies, Applied Biosystems). Relative mtDNA content was determined by normalizing Mt-Co1 expression values to Ndufv1 and calculated using the ΔΔCt method as described recently (22.Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123424) Google Scholar). Snap-frozen tissue was homogenized in ice-cold homogenization buffer (0.25 M sucrose, 1 mM EDTA, 1 mM DTT) containing protease inhibitors (20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7.0) and a phosphatase inhibitor cocktail (Sigma-Aldrich) using a handheld dispenser (Ultra-Turrax). Samples were centrifuged for 30 min at 10,000 or 20,000 g at 4°C. For immunoblotting, specific antibodies against peIF2α and eIF2α were used and GAPDH served as a loading control (all from Cell Signaling Technology, Boston, MA). HRP-conjugated goat anti-rabbit antibody (Vector Laboratories) was used as a secondary antibody. Proteins were visualized using Amersham Hyperfilm ECL (GE Healthcare). Films were scanned (GS-8000; Bio-Rad) and signal density was determined with the Quantity One® program (all from Bio-Rad Laboratories). The target protein signal was normalized to GAPDH and the fold difference of wild-type to Atgl-ko samples was determined. Data are expressed as mean ± SEM. Data were tested on normality prior to statistical analysis. Statistical significance was determined between two groups with unpaired two-tailed Student's t-test when normally distributed. Mann-Whitney test was performed when data did not show normality. Statistical significance of more than two groups was analyzed using one-way ANOVA. Group differences were considered significant for *P < 0.05, **P < 0.01, and ***P < 0.001. All statistical analyses were performed with Prism 6.0 (GraphPad Software Inc., La Jolla, CA). All raw data of the mean adipocyte area and CLS density of the experimental mouse models examined in the present study are reported in supplemental Table S1. Ambient temperature has a profound influence on body metabolism and energy expenditure, importantly affecting BAT morphology and thermogenesis (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar, 14.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar, 24.Frontini A. Cinti S. Distribution and development of brown adipocytes in the murine and human adipose organ.Cell Metab. 2010; 11: 253-256Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The impact of ambient temperature on BAT macrophage infiltration was assessed in interscapular BAT (iBAT) and mediastinal BAT (mBAT) from lean C57Bl/6j (B6) mice acclimated to cold (6°C) and warm (28°C) temperatures. In cold-acclimated mice, iBAT and mBAT were composed of typical multilocular adipocytes that exhibited a high degree of UCP-1 immunoreactivity (data not shown). MAC-2 immunohistochemistry demonstrated the absence of CLSs in iBAT from cold-acclimated mice (Fig. 1A, H) and their occasional presence in mBAT (Fig. 1C, H). Brown fat cells from lean mice kept at 28°C, a temperature close to thermoneutrality for rodents, acquired a white-like unilocular adipocyte phenotype. Notably, brown-to-white conversion did not involve significant changes in cell size either in iBAT or in mBAT (Fig. 1G). In contrast, brown-to-white conversion involved increased macrophage infiltration, which led to formation of several CLSs in iBAT (Fig. 1B, H); the density of MAC-2-positive CLSs in mBAT was also increased, even though the difference compared with cold-acclimated mice was not significant (Fig. 1D, H). To establish whether the increased CLS density seen in thermoneutral conditions was confined to BAT, inguinal WAT (iWAT) sections from cold- and warm-acclimated animals were examined. Very few CLSs were detected in iWAT from cold-acclimated mice (Fig. 1E, H), whereas in warm-acclimated animals, there was a significant increase in adipocyte area (Fig. 1G) that was, however, not matched by a significant increase in CLS density (Fig. 1F, H). These data suggest that brown adipocytes converting to a white-like unilocular phenotype at high ambient temperatures have a limited ability to store lipids and grow, and that they become more prone to death and to be cleared by CLSs compared with white adipocytes. In obese mice, brown adipocytes display a white-like appearance similar to the phenotype found in warm-acclimated mice (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar, 24.Frontini A. Cinti S. Distribution and development of brown adipocytes in the murine and human adipose organ.Cell Metab. 2010; 11: 253-256Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). We therefore investigated to determine whether iBAT from leptin receptor-deficient db/db mice showed signs of whitening and CLS formation like warm-acclimated mouse iBAT. Typical brown adipocytes were detected in db/+ mice (Fig. 2A), whereas in db/db mice they showed a white-like appearance and a significant size increase (Fig. 2B, E). CLSs were never detected in iBAT from control animals (Fig. 2A, E), whereas they were frequently observed in iBAT from db/db mice (Fig. 2B, E). BAT-to-WAT conversion in warm-acclimated and obese mice is held to be mediated by low activity of the sympathetic nervous system (13.Cinti S. The Adipose Organ.Kurtis, Milan, Italy. 1999; Google Scholar, 14.Cannon B. Nedergaard J. Brown adipose tissue: function and physiological significance.Physiol. 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