Human BAT possesses molecular signatures that resemble beige/brite cells

Abstract

Brown adipose tissue (BAT) dissipates chemical energy and generates heat to protect animals from cold and obesity. Rodents possess two types of UCP-1 positive brown adipocytes arising from distinct developmental lineages: "classical" brown adipocytes develop during the prenatal stage whereas "beige" or "brite" cells that reside in white adipose tissue (WAT) develop during the postnatal stage in response to chronic cold or PPARγ agonists. Beige cells' inducible characteristics make them a promising therapeutic target for obesity treatment, however, the relevance of this cell type in humans remains unknown. In the present study, we determined the gene signatures that were unique to classical brown adipocytes and to beige cells induced by a specific PPARγ agonist rosiglitazone in mice. Subsequently we applied the transcriptional data to humans and examined the molecular signatures of human BAT isolated from multiple adipose depots. To our surprise, nearly all the human BAT abundantly expressed beige cell-selective genes, but the expression of classical brown fat-selective genes were nearly undetectable. Interestingly, expression of known brown fat-selective genes such as PRDM16 was strongly correlated with that of the newly identified beige cell-selective genes, but not with that of classical brown fat-selective genes. Furthermore, histological analyses showed that a new beige cell marker, CITED1, was selectively expressed in the UCP1-positive beige cells as well as in human BAT. These data indicate that human BAT may be primary composed of beige/brite cells.

Figure 1. Distinct molecular signatures in Myf-5 derived classical brown adipocytes and non-Myf-5 derived beige cells.

(A) Microarray analysis was performed in fully differentiated brown adipocytes from interscapular BAT (classical brown adipocytes), white adipocytes isolated from inguinal WAT and rosiglitazone-treated adipocytes (beige cells). The color scale shows the fold changes in mRNA expression levels of the genes in blue (down-regulation)-white-red (up-regulation) scheme. (B) mRNA levels for the indicated genes were analyzed by qRT-PCR. Relative expression levels in the brown adipocyte were set as 1.0. * P<0.05, **P<0.01. Data are expressed as means ± SD.

Figure 2. Isolation of human BAT from multiple BAT depots.

(A) Axial (top) and coronal (bottom) MRI scanning images illustrating bilateral supraclavicular BAT depots from chemical-shift water-fat MRI in a 4-months-old female. BAT depots are denoted by arrows and subcutaneous WAT depots are denoted by arrowheads. The first column shows the water images. The second column shows the fat images. The third column shows the fat fraction map of water content, which translates to a lower fat fraction in BAT than WAT. The coronal images were obtained anteriorly at the level of the humeral epiphysis. (B) The isolated BAT depots were fixed and stained by H&E. The sample was obtained from the left supraclavicular area of the same 4-month old female shown in the MR images in Fig. 2A. Note that nearly all the isolated BAT samples consisted of brown adipocytes containing multilocular lipid droplets.

Figure 3. Transcriptional analyses of human BAT.

(A) Expression profiles of the common brown fat-selective genes (Group A), classical brown fat-selective genes (Group B), and beige cell-selective genes (Group C) in human BAT from multiple adipose depots. The color scale shows the mRNA levels of the genes in green (low or no-expression)-black-red (high-expression) scheme. All genes are shown in the identical scale. WAT: white adipose tissue. M: smooth muscle. Each alphabet indicates the tissues from the same patient. (B) Correlation matrix of beige-selective and classical brown-selective gene expression. The color scale shows the Pearson correlation between each gene mRNA expression in blue (negative correlation, −1.0)-gray (no correlation, 0)-red (positive correlation, 1.0) scheme. (C) Strength of correlation with representative group A gene (PRDM16 and PGC1a) and classical-brown-selective genes or beige cell-selective genes. **P<0.01 relative to representative group B gene. (D) Correlation between the mRNA expression of PRDM16 and PGCa1 (left), CITED1 (middle), and ZIC1 (right). These genes represent each gene class. Dotted lines denote density ellipse (95% confidence interval of plot). (E) Box-and-whisker plot (upper) and histogram (bottom) to graphically summarize the distribution of each gene expression levels. The lines extending from end of the box (quartile) are whiskers, which edge is the outermost data point(s) that fall within the distance defined by quartile ±1.5*(IQR). Values beyond the whiskers, denoted by a red point indicate outlier samples.

Figure 4. Expression of a new beige cell marker CITED1 in mice and in humans.

(A) Immunohistochemistry of UCP1 (left) and CITED1 (right) in serial sections of WAT in mice that were treated with CL316243 at a dose of 1 mg/kg (upper panels) or saline control (lower panels) for 8 days. Scale bar, 50 µm. (B) Immunohistochemistry of UCP1 (left) and CITED1 (right) in serial sections of human BAT. Lower panels show IgG negative controls of immunohistochemistry for UCP1 (Cy3) and for CITED1 (FITC), respectively in human BAT. Scale bar, 50 µm.