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Comparative Study
. 2014 Apr 15;306(8):E945-64.
doi: 10.1152/ajpendo.00473.2013. Epub 2014 Feb 18.

Brown and White Adipose Tissues: Intrinsic Differences in Gene Expression and Response to Cold Exposure in Mice

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Free PMC article
Comparative Study

Brown and White Adipose Tissues: Intrinsic Differences in Gene Expression and Response to Cold Exposure in Mice

Meritxell Rosell et al. Am J Physiol Endocrinol Metab. .
Free PMC article

Abstract

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Keywords: adipokine; brite; brown adipose tissue; cold; subcutaneous white adipose tissue; visceral white adipose tissue.

Figures

Fig. 1.
Fig. 1.
Global expression analysis of brown adipose tissue (BAT) and subcutaneous and mesenteric white adipose tissue (WAT) in mice exposed to cold. A: global mRNA expression was measured in BAT and subcutaneous and mesenteric WAT depots of mice exposed at either 28 or 6°C using Affymetix 2.0 ST microarrays. A row-centred heat map of hierarchical clustering carried out on the differentially expressed gene probes at a 5% false discovery rate is shown. Probe sets are colored according to the average expression level across all samples, with green denoting a lower expression level and red denoting a higher expression level. B: top pathways that were altered by cold exposure in BAT and subcutaneous WAT. The bars represent significance, and the ratio of genes altered out of the total genes per pathway is indicated. Pathway analysis was performed using up- and downregulated probes separately. mTOR, mammalian target of rapamycin; EIF, eukaryotic initiation factor; NRF2, nuclear factor-E2-related factor; PI3K, phosphatidylinositol 3-kinase; CNTF, ciliary neurotrophic factor; RAR, retinoic acid receptor.
Fig. 2.
Fig. 2.
Global expression comparison of BAT and subcutaneous (sWAT) and mesenteric WAT (mWAT) at 28°C. A: global mRNA expression was measured in BAT, sWAT, and mWAT depots of mice housed at 28°C, using Affymetix 2.0 ST microarrays. Shown is a row-centred heat map of hierarchical clustering carried out on the differentially expressed gene probes at a 10% false discovery rate. Probe sets are colored according to the average expression level across all samples, with green denoting a lower expression level and red denoting a higher expression level. B: top pathways that are enriched in each of the different depots compared with each other. The bars represent significance, whereas ratio shows the genes altered out of the total genes per pathway. Pathway analysis was performed using up- and downregulated probes separately.
Fig. 3.
Fig. 3.
Expression of selected genes induced by cold exposure or by a β3-adrenergic-specific agonist in different adipose depots. A: mRNA expression of uncoupling protein 1 (UCP1) and cell death-inducing DNA fragmentation factor α-subunit-like effector A (Cidea) in BAT, sWAT, gonadal WAT (gWAT), and mWAT of mice exposed to 28, 22, or 6°C. B: hematoxylin and eosin staining (H & E) and UCP1 immunohistochemistry. UCP1-immunoreactive brown adipocytes interspersed among unilocular white adipocytes of subcutaneous adipose tissue of mice exposed at 28 or 6°C. C: mRNA expression of genes identified by microarray in BAT, sWAT, gWAT, and mWAT of mice exposed to 28, 22, or 6°C. D: mRNA expression of selected genes in different adipose depots of mice injected intraperitoneally with the selective β3-receptor agonist CL-316,243 (1 mg/kg body wt) after 5 h; n = 4. All mRNA data are expressed as fold induction compared with BAT of mice either at 28°C or injected with saline. Bars represent the means ± SE of at least 3 mice/group, and significant differences are shown. *P < 0.05; **P < 0.005; ***P < 0.001.
Fig. 4.
Fig. 4.
Adipocyte-selective expression of temperature-regulated genes. mRNA expression of in cells from the stromal vascular fraction (SVF) or mature adipocytes prepared from interscapular BAT, sWAT, gWAT (Gon), and mWAT. Perilipin protein levels from gWAT are shown as an indicator of the quality of the fractionation (top right). Nrg, neuregulin; Nnat, neuronatin; Cil6l, cold-induced lymphocyte antigen 6-like; Letm1, leucine zipper EF hand-containing transmembrane protein 1.
Fig. 5.
Fig. 5.
Immortalized cell lines from different adipose tissue depots. A: Oil Red O staining of differentiated cells on days 8–10 of differentiation. B: mRNA expression of UCP1, PPARγ, and preadipocyte factor (Pref-1; n = 3) and a representative example of Western blot for UCP1, CIDEA, and aP2. C: mRNA expression in cells from different immortalized cell lines on days 0, 2, and 8 of differentiation. Data are expressed as fold induction compared with BAT cells on day 0; n = 3. D: mRNA expression in differentiated cells from the different immortalized cell lines that had been treated for 5 h with β3-receptor agonist CL-316,243 (10 μM) or vehicle control (Ctrl). Data are expressed as fold induction compared with nonstimulated BAT cells; n = 3. For all of the mRNA data, bars represent means ± SE, and significant differences are shown. *P < 0.05; **P < 0.005; ***P < 0.001. PGC-1α, PPARγ coactivator-1α.
Fig. 6.
Fig. 6.
Functional characterization of immortalized cell lines from different adipose tissue depots. A: [3H]palmitate oxidation in differentiated adipocytes generated from interscapular BAT and sWAT, gWAT, and mWAT treated with or without CL-316,243 (CL; 10 μM) for 5 h. Results are expressed as a mean of 3 experiments and radioactivity count numbers normalized to DNA content. B: basal, insulin (100 nM), and CL (10 μM, 5 h) stimulated glucose uptake measured with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose. Results are expressed as a mean of 3 experiments and fluorescence normalized to protein content. p-Akt was measured by Western blot with antibody against phosphorylated Ser473. A representative blot is shown. GLUT1 and GLUT4 mRNA relative expression levels are shown in differentiated cells. C: IL-6 production by differentiated adipocytes after 24-h treatment with increasing amounts of LPS. Bars represent means ± SE, and significant differences are shown. *P < 0.05; **P < 0.01.
Fig. 7.
Fig. 7.
Motif analysis of brite gene promoters. Motif logos are presented from the MEME-LaB-analyzed gene cluster for transcripts induced by cold in subcutaneous WAT and more highly expressed in BAT vs. WAT depots. The MEME-LaB software determined 10 motifs, and these were analyzed for similarity to know motifs from JASPAR, PLACE, and TRANSFAC using position-specific scoring matrix (PSSM). Eukaryotic transcription factor binding sites are indicated for each relevant PSSM.
Fig. 8.
Fig. 8.
Nrg4 is a BAT adipokine promoting neurite outgrowth. A: Nrg4 mRNA expression in mouse tissues (n = 4). Data are expressed as a fold difference compared with kidney. B: inmunohistochemistry of Nrg4 in murine BAT and gonadal WAT. C: Nrg4 and aP2 mRNA expression in immortalized BAT cells throughout differentiation. Data are shown as fold induction compared with time 0. D: NRG4 levels in unconditioned medium (control), conditioned medium from undifferentiated brown adipocytes (Undiff Cond), and 4-wk-differentiated brown adipocytes (Diff Cond) in 6-well plates, determined by ELISA. DNA content was 28.65 ± 1.38 and 26.04 ± 1.96 μg/well for differentiated and undifferentiated cells, respectively. E: neurite staining with anti-Tuj1 antibody of PC12-HER4 cells incubated with different concentrations of conditioned medium from differentiated brown adipocytes. Neurite outgrowth measured in μm and expressed as fold induction over control cells. F: neurite staining by anti-Tuj1 antibody of PC12-HER4 treated with conditioned medium of differentiated brown adipocytes stably expressing shNrg4 or a nontargeting shRNA. For all of the data, bars represent means ± SE, and significant differences are shown. *P < 0.05.

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References

    1. Aguilo F, Camarero N, Relat J, Marrero PF, Haro D. Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma. Biochem J 427: 255–264, 2010 - PubMed
    1. Aissani B, Perusse L, Lapointe G, Chagnon YC, Bouchard L, Walts B, Bouchard C. A quantitative trait locus for body fat on chromosome 1q43 in French Canadians: linkage and association studies. Obesity (Silver Spring) 14: 1605–1615, 2006 - PubMed
    1. Almind K, Manieri M, Sivitz WI, Cinti S, Kahn CR. Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice. Proc Natl Acad Sci USA 104: 2366–2371, 2007 - PMC - PubMed
    1. Anderson GR, Posokhova E, Martemyanov KA. The R7 RGS protein family: multi-subunit regulators of neuronal G protein signaling. Cell Biochem Biophys 54: 33–46, 2009 - PMC - PubMed
    1. Asano A, Irie Y, Saito M. Isoform-specific regulation of vascular endothelial growth factor (VEGF) family mRNA expression in cultured mouse brown adipocytes. Mol Cell Endocrinol 174: 71–76, 2001 - PubMed

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