Identification and characterization of distinct brown adipocyte subtypes in C57BL/6J mice

(A) Quantification of relative lipid accumulation measured by Oil Red O staining at day 8 of differentiation from 67 immortalized brown preadipocyte clones (n = 4–6; mean OD normalized to DAPI ± SEM). (B) Representative images of differentiated cell lines, stained with F-actin (red), lipid droplets (green), and DAPI (blue). (C) Correlation between mean lipid droplet size and lipid area normalized by the number of nuclei per clone (n > 200), calculated from pictures shown in Supplemental Data 2. Values are mean of different area scanned per clones (n = 9). (D) Correlation analysis of Pparg expression and lipid accumulation. The values were mean ± SEM (n = 5).

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(A) mRNA expressions of Ucp1 at day 8 of differentiation (n = 2–5). (B) Heat map of mRNA expression for different adipocyte markers in all 67 clones, and in vitro differentiated white, beige and brown adipocytes at day 8 of differentiation (n = 3–8). Median expression for each cell line was transformed to log scale and gene wise z-scores were computed independently for each cell line. Dendrogram colors denote identified adipocyte clone cluster. (C) Correlation of Ucp1 with Pparg (left panel), Prdm16 (middle panel), and Ppargc1a (right panel). The values were mean ± SEM, and log transformed for Ucp1, Prdm16, and Ppargc1a (n = 4–5). Gene expression was normalized to Tbp. (D) Ucp1 expression of controls and cells treated for 3 h with 0.5 μM CL-316,243 (n = 2–5).

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Transcriptional profiling of selected BAT clones. (A) Heat map of all 9,483 expressed genes in preadipocyte. Gene expression in rows was z-score normalized, columns refer to cell lines (left panel). Hierarchical clustering heat map of all 10,363 expressed genes in differentiated adipocytes (right panel). (B) PCA plot of the first two PC of preadipocytes expression data (upper panel) and differentiated adipocytes (lower panel). (C) Scatterplot comparison of the estimated brown adipose tissueness from pre- and differentiated adipocyte cell lines. Dots, triangles, and square indicate the three clusters identified by k-means clustering.

(A) Brown preadipocyte gene expression in Laplacian eigenmap–derived low dimensional space. Expression of the three eigenvectors ϕ2, ϕ4, and ϕ6. Distance from estimated center is color-coded (left panel). After removal of genes with low variance module membership is coded in colors (right panel). (B) Heat map of hierarchical clustered module genes in rows. Gene expression was z-score normalized. Module membership is indicated by the right color bar. (C) Distribution of correlation coefficients of the gene-wise comparison of expression to estimated brown adipose tissue (BAT)ness for each module. Upper line: preadipocyte gene expressions versus estimated BATness of preadipocytes from ProFat database. Middle line: preadipocyte expressions versus differentiated BATness from ProFat database. Lower line: differentiated adipocyte expressions versus differentiated BATness from ProFat database. (D) Network of KEGG-enriched pathways. Significantly enriched pathways are connected to the respective module nodes. Size of the pathway nodes and proportion of the node-pie-chart refer to −log₁₀ of enrichment P-value. Color of the nodes refers to module membership.

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(A) Heat maps of stably expressed genes of preadipocytes (left panel) and differentiated brown adipocytes (right panel). Module membership is indicated by the right color bar. (B) Correlation coefficients for stable expressed genes compared with estimated brown adipose tissue (BAT)ness. Color of bars indicates module membership. (C) Correlation plot for pairwise comparison of selected markers to selected stably expressed genes. Red plots denote a significant correlation. (D) Expression of Ucp1, Eif5, Tcf25, and Bin1 in mice kept in cold or thermoneutrality (n = 6–8, data are mean expressions normalized to B2m ± SEM). (E) Expression analysis of Ucp1, Eif5, Tcf25, and Bin1 in chow- and high-fat diet-fed mice (n = 4–6, data were mean expressions normalized to B2m ± SEM). (F) BAT co-staining for EIF5, TCF25 or BIN1 (red) and UCP1 (green), with F-actin (gray) and Dapi (blue) from wild-type C57BL/6J mice. Arrows indicate nuclear staining. (G) Representative color gradient picture of EIF5 staining on BAT (left panel), and percentage area of different EIF5 intensity (high, medium, low, and very low) normalized to total area (second panel, n = 8 sections). Quantification of TCF25- and BIN1-positive and negative cells in percentage of total cells (n = 9–18 sections). (H) Heat map of mRNA expression in human periadrenal (ADR), supraclavicular (SCLV), and subcutaneous (SCT) adipose tissue from six different donors for ADR and SCLV, and five donors for SCT. (A, B) Based on RNAseq data and remaining analysis based on RT-qPCR data. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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(A) Heat map and hierarchical clustering of cell lines with regard to the mRNA expressions of Eif5, Tcf25 and Bin1. (B) Expression of Eif5, Tcf25, and Bin1 in control cells (shScr) of different clones (B1, D1, and D5) and the respective knockdown (shEif5 B1, shTcf25 D1, and shBin1 D5); Preads (d0 of differentiation) and Ads (d8 of differentiation) (n = 5). (C) Oxygen consumption rate of shScr B1 and shEif5 B1 (left panel), shScr D1, and shTcf25 D1 (middle panel) and shScr D5 and shBin1 D5 (right panel) at day 8 of differentiation measured by Seahorse (n = 4–5). (D) Expression of Eif5, Tcf25, and Bin1 in control cells (shScr) and respective knock-down (shEif5, shTcf25, and shBin1); Preads (d0 of differentiation) and Ads (d8 of differentiation) (n = 5). (E) Immunofluorescence staining of EIF5/TCF25/BIN1 (green), F-actin (red), and DAPI (blue) on shScr/shEif5/shTcf25/shBin1 preadipocytes. (F) Representative images of differentiated cell lines (shScr, shEif5, shTcf25, and shBin1) stained with F-actin (white), lipid droplets (red), and DAPI (blue). (G) Quantification of relative lipid accumulation measured by Oil Red O staining at day 8 of differentiation from knockdown cell lines (n = 4, mean OD normalized to DAPI ± SEM). (H) mRNA expressions of Pparg (left panel) and Ucp1 (right panel) on shScr, shEif5, shTcf25, and shBin1 adipocytes and CL-316,243-treated adipocytes (0.5 μM CL-316,243 for 3 h; n = 6). (I) Quantification of Western blots for UCP1 (Cat. no. 10983; Abcam) in shScr, shEif5, shTcf25, and shBin1 adipocytes and adipocytes treated with 0.5 μM CL-316,243 for 6 h (n = 6–7) normalized to β-actin. Values are fold changes to the respective shScr adipocytes (ads). (J) Oxygen consumption rate of control and knockdown cell lines at day 8 of differentiation measured by Seahorse (n = 3); data were normalized by non-mitochondrial respiration. (K) Schematic illustration of brown adipose tissue heterogeneity: Murine brown adipose tissue is composed of functionally distinct brown adipocytes. We identify EIF5 expressing brown adipocytes as “classical” brown adipocytes, with high UCP1 content and mitochondrial uncoupling. TCF25 expressing brown adipocytes are similar, albeit with lower UCP1 expression. In contrast, BIN1-expressing brown adipocytes appear in a dormant state, expressing low UCP1 levels with little response to beta adrenergic stimulation. However, loss of BIN1 or chemical mitochondrial uncoupling reveals high thermogenic capacity of these cells. Subpopulations of brown adipocytes are color coded regarding to their thermogenic capacity, with the highest shown in dark brown (Eif5^high), followed by brown (Tcf25^high) and light brown (Bin1^high) (=indicates unchanged expression). All RT-qPCR data were normalized to Tbp and shown as mean ± SEM. *P < 0.05, **, ^##P < 0.01, ***P < 0.001, ****P < 0.0001.

Source data are available for this figure.