Activation of Classical Brown Adipocytes in the Adult Human Perirenal Depot Is Highly Correlated with PRDM16–EHMT1 Complex Expression

<div><p>Brown fat generates heat to protect against cold and obesity. Adrenergic stimulation activates the thermogenic program of brown adipocytes. Although the bioactivity of brown adipose tissue in adult humans had been assumed to very low, several studies using positron emission tomography–computed tomography (PET–CT) have detected bioactive brown adipose tissue in adult humans under cold exposure. In this study, we collected adipose tissues obtained from the perirenal regions of adult patients with pheochromocytoma (PHEO) or non-functioning adrenal tumors (NF). We demonstrated that perirenal brown adipocytes were activated in adult patients with PHEO. These cells had the molecular characteristics of classical brown fat rather than those of beige/brite fat. Expression of brown adipose tissue markers such as uncoupling protein 1 (UCP1) and cell death-inducing DFFA-like effector A (CIDEA) was highly correlated with the amounts of PRD1-BF-1-RIZ1 homologous domain-containing protein-16 (PRDM16) – euchromatic histone-lysine N-methyltransferase 1 (EHMT1) complex, the key transcriptional switch for brown fat development. These results provide novel insights into the reconstruction of human brown adipocytes and their therapeutic application against obesity and its complications such as type 2 diabetes.</p></div

Brown adipose tissues are activated in the perirenal regions of PHEO patients.

<p>Comparisons of (A–G) BAT-associated mRNA (A, <i>UCP1</i>; B, <i>PPARGC1A</i>; C, <i>CIDEA</i>; D, <i>ELOVL3</i>) and protein (F, UCP1; G, CIDEA) between NF and PHEO in adipose tissues from the perirenal regions. (E) Protein quantification was performed using western blot analysis. (H) H&E staining (top) and immunohistochemistry using antibodies against UCP1 (middle) and CIDEA (bottom). Scale bar, 50 μm. *<i>P</i> < 0.05; **<i>P</i> < 0.01; NS, not significant.</p

Subject characteristics.

<p>Comparisons between the NF and PHEO groups.</p><p>Values are the means ± SEM, median (interquartile range), number, or <i>P</i>-value.</p><p>*Values were normalized by logarithmic conversion before comparisons.</p><p>Subject characteristics.</p

The PRDM16–EHMT1 complex plays an important role in the adult human brown adipose cell development.

<p>(A, B) Correlation analyses at the mRNA level were performed in tissues from patients with PHEO between <i>PRDM16</i> (A) or <i>EHMT1</i> (B) and BAT-associated genes (left to right, <i>UCP1</i>, <i>PPARGC1A</i>, <i>CIDEA</i>, and <i>ELOVL3</i>). (C, D) The correlation was also assessed at the protein level between PRDM16 and UCP1 (C, left), PRDM16 and CIDEA (C, right), EHMT1 and UCP1 (D, left), or EHMT1 and CIDEA (D, right). Protein expression levels were assessed by western blot analysis (shown in Fig. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122584#pone.0122584.g001" target="_blank">1E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122584#pone.0122584.g003" target="_blank">3E</a>). (F) Correlation between the PRDM16 protein and EHMT1 protein in PHEO samples. The protein expression levels were assessed by western blot analysis (E).</p

The expression levels of BAT-associated genes are not affected by clinical characteristics.

<p>(A) The correlation matrix between BAT-associated gene expression (top to bottom: UCP1, CIDEA, PPARGC1A, or ELOVL3) and subject characteristics (left to right: age, gender, body mass index (BMI), and total preoperative urinary catecholamine levels) in patients with NF (cross) or PHEO (circle). (B) Correlation analysis between individual urinary catecholamines and <i>UCP1</i> mRNA. *Values were normalized by logarithmic transformation before each correlation analysis.</p

Activated BAT in the perirenal region has the molecular signatures of classical brown fat.

<p>(A) The heat map matrix of gene expression profiles in PHEO subjects. Subjects are arranged in order of <i>UCP1</i> mRNA expression levels. All data are normalized within each column using the Z-score method. The color scale shows the Z-score; the highest value is bright red, the lowest is bright blue, and the midpoint is black. (C) Comparison of EBF3 protein expression levels between NF and PHEO subjects in perirenal adipose tissues. The protein expression levels were assessed by western blot analysis (B). (D) Representative example of immunostaining for EBF3 in a PHEO sample. Scale bar, 50 μm. *<i>P</i> < 0.05; **<i>P</i> < 0.001, NS, not significant.</p