(A) Multilocular beige fat cells are prominent in the subcutaneous white adipose depot of 129SVE male mice at 7–9 weeks of age. Hematoxylin & Eosin stain show islets of multilocular fat cells within inguinal white fat depot (400X). (B) Total RNA was isolated from epididymal (Epi), inguinal (Ing) and interscapular (Bat) adipose tissues of 129SVE mice and assayed for mRNA expression for Ucp-1 and other brown fat-like genes by qPCR. Values are mean±SD (n=5). Expression levels of Ucp-1 and Cidea are undetected in epididymal depot. (C) Total RNA was isolated from fat cells differentiated from epididymal (Epi), inguinal (Ing) and interscapular (Bat) primary stromal vascular cells and assayed for mRNA expression for Ucp-1 and other brown fat-like genes by qPCR. Relative gene expression was normalized to adipsin mRNA levels. Values are mean±SD (n=3) (D) Cluster dendrogram of array expression data with RNA samples from differentiated cultures of 26 immortalized fat cell lines as described in text. Analysis details were described in the . Height (y axis) is Euclidean distance. To mimic the sympathetic tone in vivo upon prolonged cold exposure when beige fat cells are functionally activated, we analyzed RNA from fully differentiated cells (day8), treated with 10 μM forskolin (FSK), a cAMP inducing agent, for 4 hrs.

Representative lines from each group were further analyzed functionally. Bat1, bat6, bat8 for brown fat cells; X9, X3, D16, D12 for beige fat cells and N13, X7, J6, G18 for white fat cells. All these lines presented similar levels of adipogenesis and fat cell specific markers were measured via qPCR. See . Similar results were observed in more than three independent experiments. (A) Total RNA was isolated from the differentiated brown, beige and white clonal cell lines and was assayed for mRNA levels of brown fat-like genes (Ucp-1, Cidea and Cox7a1) by qPCR. (B) Total RNA was isolated from the differentiated brown, beige and white clonal cell lines treated with 10μM forskolin for 4 hours and was assayed for mRNA levels of Ucp-1. (C) Protein lysates were isolated from the differentiated brown, beige and white clonal lines either unstimulated or treated with 10 μM isoproterenol for 6 hours, and probed by immunoblot as indicated, with tubulin as a loading control. (D) Cell transplantations were done in the preadipose state as described in Experimental Procedures. After 6 weeks, fat pads were harvested and mRNA expression was analyzed by qPCR (n=5–8). For acute sympathetic stimulation, CL316,243, at 1mg kg⁻¹, was injected intraperitoneally 5 hours before harvesting the transplanted fat pads. (E) Oxygen consumption in cultured brown, beige and white fat cells was assayed as described in Experimental Procedures. Oligomycin (ATP synthase inhibitor) was added to cells to measure the uncoupled respiration rate. The cells were treated with dibutyryl-cAMP for 12 hours before harvesting. Values are mean±SD (n=4). *, p<0.05; **, p<0.01; ***, p<0.001.

(A) Genes with a beige- or brown-selective expression pattern were first identified by microarray analysis as described in . Relative mRNA expression for these genes was then measured by qPCR in differentiated brown, beige and white fat cells in an unstimulated state. (B) Analysis of beige- and brown-selective genes expression in adipose tissues isolated from inguinal WAT and interscapular BAT of 129 SVE mice. Values are mean±SD (n=5). *, p<0.05; **, p<0.01; ***, p<0.001. (C) Protein lysates of adipose tissues isolated from inguinal WAT and interscapular BAT of 129 SVE mice were probed with antibodies by immunoblot as indicated. Asterisk represents a nonspecific band as a loading control. (D) Microscopic and confocol images of mouse inguinal white and interscapular brown adipose tissue stained with antibodies recognizing CD137 (red) (microscopic images), TMEM26 (green) (confocal images) and UCP1 (green and red), with nuclei co-stained with DAPI as indicated. To induce the “browning” morphology and UCP1 expression in inguinal white adipose depot, 129SVE mice were injected intraperitoneally with CL316, 243, at 1mg kg⁻¹ daily for 4 days. Inguinal and interscapular adipose tissues were harvested on the fifth day.

Total RNA was isolated from differentiated cultures of beige-selective marker CD137 (A) and TMEM26 (B) positive and negative cells purified from inguinal SV cells; these adipocytes were then assayed for mRNA levels of general adipogenic markers, Ucp-1 and other characteristic thermogenic/brown fat-like genes by qPCR. (C) Primary inguinal SV cells were purified as in (A) and differentiated into adipocytes for 6 days in the presence of FC fragment of human IgG (hFC) (100nM), fusion protein of irisin-FC (100nM) or recombinant FNDC5 (20nM). Total RNA was isolated and assayed for mRNA levels of general adipogenic marker aP2, thermogenic gene Ucp-1 and other brown-like genes by qPCR. Values are mean±SD (n=3). *, p<0.05, **, p<0.01, ***, p<0.001. Similar results were observed in more than three independent experiments.

Expression of beige- and brown-selective genes in hWAT versus hBAT samples from total 11 subjects of two independent cohorts were measured by qPCR. (A) UCP1 expression levels were measured and confirmed to be higher in BAT as compared to WAT. (B) Heatmap summary of relative fold changes in expression of these genes in each subject. Relative gene expression level of 3 beige-selective (C), 3 brown-selective (D) are shown individually. P value was analyzed using Wilcoxom matched pairs signed ranks test.

Microscopic images of adult supraclavicular adipose tissue stained with antibodies recognizing CD137 (red, A and C), UCP1 (green, A), TMEM26 (green, B) and perilipin (green, C) as indicated with nuclei stained with DAPI. It is worth noting that in (A) and (B) the expression of both CD137 and TMEM26 in multilocular (see the arrowhead in B), UCP1+ adipocytes (labeled with “BAT”), but the neighboring perilipin-1+, UCP1− adipocytes are CD137− (labeled with “WAT”, see C).