Androgen Reduces Mitochondrial Respiration in Mouse Brown Adipocytes: A Model for Disordered Energy Balance in Polycystic Ovary Syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrinopathy that is associated with an adverse metabolic profile including reduced postprandial thermogenesis. Although abnormalities in adipose tissue function have been widely reported in women with PCOS, less is known about direct effects of androgen on white and, particularly, brown adipocytes. The purpose of this study was to investigate the effect of the nonaromatizable androgen dihydrotestosterone (DHT) on (1) lipid accumulation and expression of adipogenic markers in immortalized mouse brown adipose cell lines (IMBATs), (2) mitochondrial respiration in IMBATs, (3) mitochondrial DNA content and gene expression, (4) expression of brown adipose tissue (BAT) markers and thermogenic activation. In addition, we profiled the relative levels of 38 adipokines secreted from BAT explants and looked at androgen effects on adipokine gene expression in both IMBATs and immortalized mouse white adipose (IMWATs) cell lines. Androgen treatment inhibited IMBAT differentiation in a dose-dependent manner, reduced markers of adipogenesis, and attenuated the β-adrenoceptor-stimulated increase in uncoupling protein-1 (UCP1) expression. In explants of mouse interscapular BAT, androgen reduced expression of UCP1, peroxisome proliferator-activated receptor-γ coactivator-1 (PCG-1) and Cidea. Significantly, as well as affecting genes involved in thermogenesis in BAT, androgen treatment reduced mitochondrial respiration in IMBATs, as measured by the Seahorse XF method. The results of this study suggest a role for excess androgen in inhibiting brown adipogenesis, attenuating the activation of thermogenesis and reducing mitochondrial respiration in BAT. Together, these data provide a plausible molecular mechanism that may contribute to reduced postprandial thermogenesis and the tendency to obesity in women with PCOS.

Keywords: PCOS; UCP1; androgens; brown adipose tissue; mitochondrial respiration.

Figure 1

Dihydrotestosterone (DHT) treatment reduces brown adipogenesis. (A) Representative images showing presence of the androgen receptor protein in brown preadipocytes and adipocytes. Blue—nuclear stain DAPI, green—androgen receptor. (B) Representative differential interference contrast (DIC) microscopy images showing a reduction of lipid accumulation and brown adipocyte differentiation with increasing dose of DHT. (C) Representative images showing a reduction in lipid droplet formation with DHT, measured by Oil Red O in control and the 10 μM DHT. (D) Relative mRNA expression of adipogenic markers in brown adipocytes differentiated for seven days in the presence of different doses of DHT (t-test, * p < 0.05. Data shown is mean (±SEM n = 4).

Figure 2

Reduced mitochondrial respiration in brown adipocytes treated with DHT. (A) Bioenergetic profile of brown adipocytes treated with control (black line), 100 nM DHT (red line) and 1μM DHT (blue line). (B) Oxygen consumption rate (OCR) shows reduction in both basal and maximal respiration in brown adipocytes treated with either 100 nM DHT (red bar) and 1μM DHT (blue bar). One-way ANOVA with Holm–Sidak’s multiple comparison test, * p < 0.05, ** p < 0.01. Data shown are mean (±) SEM, n = 6.

Figure 3

DHT treatment reduces UCP1 gene expression and activation of the thermogenic pathway. (A) Relative mitochondrial DNA content in brown adipose tissue treated with either control or 100 nM DHT. Representative images showing no change in mitochondrial number, observed by MitoTracker Red in control and the DHT treated brown adipocytes. (B) Relative mRNA expression of mitochondrial function genes in brown adipose tissue treated with either control or 100 nM DHT. (C) Relative mRNA expression of brown adipose markers in brown adipose tissue treated with either control or 100 nM DHT. (D) Thermogenic activation measured by UCP1 mRNA increase in brown adipocytes treated with either control, 1μM isoproterenol, or 1 μM isoproterenol co-treated with 100 nM of DHT. t-test, * p < 0.05. Data shown is mean (±) SEM n = 4).

Figure 4

(A) Heat-map produced from proteome profiler adipokine arrays used to profile the levels of secreted adipokines from interscapular BAT explants cultured in control medium for 24 h. Red indicates adipokines secreted at high levels and green indicates adipokines secreted at lower levels from brown adipose tissue (n = 4). (B) Heat-map showing adipokine gene expression in BAT relative to WAT. On the scale bar, red indicates genes expressed at lower levels in BAT and green indicates genes expressed at higher levels in BAT, black represents genes that are expressed equally in BAT and WAT. (C) Relative mRNA expression of adipokine screen in IMWATs and IMBATs treated with either control of 10 μM DHT. t-test, * p < 0.05, ** p < 0.01, *** p < 0.005. Data shown are mean (±) SEM, n = 6–8.