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

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

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

Distinct Time Course of the Decrease in Hepatic AMP-Activated Protein Kinase and Akt Phosphorylation in Mice Fed a High Fat Diet

<div><p>AMP-activated protein kinase (AMPK) plays an important role in insulin resistance, which is characterized by the impairment of the insulin-Akt signaling pathway. However, the time course of the decrease in AMPK and Akt phosphorylation in the liver during the development of obesity and insulin resistance caused by feeding a high fat diet (HFD) remains controversial. Moreover, it is unclear whether the impairment of AMPK and Akt signaling pathways is reversible when changing from a HFD to a standard diet (SD). Male ddY mice were fed the SD or HFD for 3 to 28 days, or fed the HFD for 14 days, followed by the SD for 14 days. We examined the time course of the expression and phosphorylation levels of AMPK and Akt in the liver by immunoblotting. After 3 days of feeding on the HFD, mice gained body weight, resulting in an increased oil red O staining, indicative of hepatic lipid accumulation, and significantly decreased AMPK phosphorylation, in comparison with mice fed the SD. After 14 days on the HFD, systemic insulin resistance occurred and Akt phosphorylation significantly decreased. Subsequently, a change from the HFD to SD for 3 days, after 14 days on the HFD, ameliorated the impairment of AMPK and Akt phosphorylation and systemic insulin resistance. Our findings indicate that AMPK phosphorylation decreases early upon feeding a HFD and emphasizes the importance of prompt lifestyle modification for decreasing the risk of developing diabetes.</p></div

Hepatic AMPK and Akt phosphorylation levels in mice subjected to dietary change or SD.

<p>(A and B) Representative western blot quantification of AMPK phosphorylation (relative to total AMPK protein). n = 4‒5 per group. (C and D) Representative western blot quantification of insulin-stimulated Akt phosphorylation (relative to total Akt protein). n = 4‒5 per group.</p

Dietary change improves weight gain, liver steatosis, glucose tolerance, and insulin resistance.

<p>The diet was changed on day 14 (black diamonds) or continued as a SD (white circles). (A) Body weight. Results are means ± standard deviation. n = 5 per group. *<i>P</i> < 0.05. (B) Oil Red O-stained liver sections from SD-fed mice or those subjected to a dietary change. Scale bar, 100 μm. (C and D) Intraperitoneal glucose tolerance test (GTT) in SD-fed mice or those subjected to a dietary change on day 3 (C) or day 14 (D). (E and F) Insulin tolerance test (ITT) in SD-fed mice or those subjected to a dietary change on day 3 (E) or day 14 (F). Results are means ± standard deviation. n = 4‒5 per group. *<i>P</i> < 0.05.</p

Hepatic AMPK and Akt phosphorylation levels in SD- or HFD-fed mice.

<p>(A and B). Representative western blots and quantification of AMPK phosphorylation (relative to total AMPK protein). n = 4 per group. *<i>P</i> < 0.05. (C) and (D). Representative western blots and quantification of insulin-stimulated Akt phosphorylation (relative to total Akt protein). n = 4 per group. *<i>P</i> < 0.05.</p