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. 2017 Jan;31(1):266-281.
doi: 10.1096/fj.201600787RR. Epub 2016 Oct 12.

Pharmacologic activation of estrogen receptor β increases mitochondrial function, energy expenditure, and brown adipose tissue

Suriyan Ponnusamy  1 , Quynh T Tran  2 , Innocence Harvey  3 , Heather S Smallwood  4 , Thirumagal Thiyagarajan  1 , Souvik Banerjee  5 , Daniel L Johnson  6 , James T Dalton  7 , Ryan D Sullivan  8 , Duane D Miller  5 , Dave Bridges  3   4 , Ramesh Narayanan  9   10
Affiliations

Pharmacologic activation of estrogen receptor β increases mitochondrial function, energy expenditure, and brown adipose tissue

Suriyan Ponnusamy et al. FASEB J. 2017 Jan.

Abstract

Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor β (ER-β) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-β and its ligands. Estrogen receptor β (ER-β) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-β-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-β ligand were not observed in ER-β-knockout mice. Serum metabolite profiles of adult lean and juvenile mice were comparable, while that of adult obese mice was distinct, indicating a possible impact of obesity on age-dependent metabolism. This phenotype was partially reversed by ER-β-selective ligand. These data highlight a new role for ER-β in adipose biology and its potential to be a safer alternative peripheral therapeutic target for obesity.-Ponnusamy, S., Tran, Q. T., Harvey, I., Smallwood, H. S., Thiyagarajan, T., Banerjee, S., Johnson, D. L., Dalton, J. T., Sullivan, R. D., Miller, D. D., Bridges, D., Narayanan, R. Pharmacologic activation of estrogen receptor β increases mitochondrial function, energy expenditure, and brown adipose tissue.

Keywords: exercise mimetic; metabolic diseases; mitochondria; obesity; oxygen consumption.

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Figures

Figure 1.
Figure 1.
ER-β-selective ligand inhibits obesity. A) Structure of β-LGND2. B) β-LGND2 inhibits HFD-induced body weight and fat mass better than commercial drugs. Male C57BL6/J mice (6–8 wk old; n = 6–7 per group) were fed with ND or HFD. Animals fed with HFD were treated with vehicle, 30 mg/kg/d s.c. β-LGND2, lorcaserin (18 mg/kg/twice daily/s.c.), or orlistat (10 mg/kg/d s.c.). Body weight (left panel) was recorded weekly, and body fat mass (center panel) was measured using MRI once every 3 wk. Animals were humanely killed after 9 wk, and epididymal WAT weight was recorded (right panel). C) β-LGND2 inhibits body weight and fat in ob/ob mice. Male ob/ob mice (6–8 wk; n = 5) were treated with vehicle or 30 mg/kg/d s.c. β-LGND2. Weekly body weight and fat mass were recorded. Values are represented as percentage change from initiation of experiment. D) β-LGND2 inhibits body fat in WT, but not in ER-βKO, male mice. Male WT or ER-βKO mice (6–8 wk old; n = 6 per group) were maintained on ND or HFD and treated with vehicle or 30 mg/kg/d s.c. β-LGND2. Body fat was measured once every 3 wk using MRI. Animals were humanely killed, and epididymal WAT weight was recorded. Values are expressed as averages ± se. Lorc, lorcaserin; Orli, orlistat. *P < 0.05 vs. ND; #P < 0.05 vs. HFD.
Figure 3.
Figure 3.
Metabolomics in WAT indicate enhancement of mitochondrial function and energy metabolism by β-LGND2. Metabolites were profiled in WAT obtained from animals (n = 4 per group) shown in Fig. 1B. A) Heat map representing statistically different metabolites. Scale and metabolites category are provided to right of heat map. B) PCA shows clustering of individual samples used in metabolite profiling. C–E) Significantly different metabolites belonging to TCA cycle (C), oxphos (D), and glucose metabolism (E) are represented. Values are expressed as averages ± se. *P < 0.05 vs. ND; #P < 0.05 vs. HFD.
Figure 2.
Figure 2.
ER-β-selective ligand increases expression of marker genes associated with BAT in WAT. A) RNA from epididymal WAT from mice (n = 3 per group) shown in Fig. 1B was sequenced using Ion Torrent next-generation sequencer. Significantly different genes are expressed in heat map. B, C) Genes regulated by HFD (B) and β-LGND2 (C) are represented as fold change. D) Top genes regulated in HFD-fed mice compared to ND-fed mice. E) Top genes (based on P value) that were up-regulated by β-LGND2 compared to HFD vehicle-treated samples are represented. F) Genes representing BMP4 signaling pathway. Values are represented as average ± se fold change from ND vehicle-treated animals or from HFD vehicle-treated animals. *P < 0.05 vs. ND; #P < 0.05 vs. HFD.
Figure 4.
Figure 4.
ER-β inhibits preadipocyte and MSC differentiation toward adipocytes and increases BAT marker genes. A) Schematic showing experimental design adopted to differentiate preadipocytes 3T3-L1 and MSCs toward mature adipocytes. B) Overexpression of ER-β inhibits differentiation of preadipocytes. 3T3-L1 cells stably transfected with GFP, ER-α, or ER-β lentivirus were differentiated in presence of 10 nM estradiol as indicated in A. At the end of 14 d, oil droplets were imaged under a microscope. Representative image is shown. Numbers of oil droplets in 10 random fields from each transfection were quantified and are represented in right panel. C) Expression of ER-β ligand-dependently increases expression of BAT marker genes. 3T3-L1 cells were stably transfected with GFP, ER-β, or ER-α. Cells were differentiated toward mature adipocytes in presence of 10 nM estradiol for 14 d, as depicted in A. At the end of 14 d, RNA was isolated, and expression of WAT (left panel) and BAT (right panel) marker genes was quantified using real-time PCR and normalized to expression of glyceraldehyde phosphate dehydrogenase (GAPDH), and expression of ER-α and ER-β was quantified (right panel). D) ER-β inhibits differentiation of MSCs toward mature adipocytes. MSCs stably transfected with GFP or ER-β were differentiated as described in presence of 1 μM β-LGND2. At the end of 14 d, RNA was isolated, and expression of WAT (LPL-1, CEBP, and FABP4) and BAT (UCP-1 and CYC1) marker genes was quantified using real-time PCR and normalized to expression of GAPDH. E) Time course of UCP-1 induction by β-LGND2 in ER-β-expressing adipocytes. GFP- or ER-β-expressing 3T3-L1 cells were differentiated toward mature adipocytes and treated (1 μM β-LGND2) on different days of differentiation. UCP-1 expression was quantified and normalized to expression of GAPDH. Values are expressed as averages ± se. *P < 0.05 vs. ND; #P < 0.05 vs. HFD.
Figure 5.
Figure 5.
ER-β and its ligands increase oxygen consumption and mitochondrial respiration without increasing physical activity. A) Male C57BL6/J mice (6–8 wk old; n = 8 per group) fed with HFD and treated with vehicle or 30 mg/kg/d s.c. β-LGND2 were maintained in CLAMS at 25°C for entire duration of experiment. Volume of oxygen (left panel) and ambulatory activity (right panel) were measured constantly 24 h/d. HFD and drug treatment initiation time are indicated by arrow. B) Mice (n = 3 per group) described in A that were maintained at 25°C for first 15 d were exposed to cold (18°C) and continued to receive HFD and vehicle or β-LGND2 treatment. Volume of oxygen consumed was measured constantly 24 h/d. Cold exposure initiation is shown by arrow. C) Body core temperature of mice fed with HFD and treated with vehicle or β-LGND2 at 25°C and 30°C. D) Mitochondrial marker genes were up-regulated in WAT in animals maintained in cold and in animals treated with β-LGND2. RNA was isolated from WAT of animals shown in A and B. Expression of mitochondrial genes was measured using real-time PCR and normalized to large ribosomal protein (RPLPO). E) OCR and ECAR were increased by ER-β and its ligands. 3T3-L1 cells that were stably transfected with GFP or ER-β were seeded in Seahorse plates and were differentiated in presence or absence of 10 nM estradiol or 1 μM β-LGND2. Ten days after differentiation, mitochondrial respiration was measured with Seahorse Bioanalyzer (n = 4). All points on E in ER-β transfected cells were significantly different compared to GFP transfected cells. *P < 0.05 vs. ND; #P < 0.05 vs. HFD.
Figure 6.
Figure 6.
Age-related changes in serum metabolites in lean and obese mice. Metabolites were identified in serum of animals (n = 4 per group) shown in Fig. 1B. Serum was isolated from blood at euthanasia, and metabolites were identified. Baseline samples (n = 4) were also obtained before initiation of experiment for comparison with end-of-experiment samples. A) PCA shows clustering of individual samples. B) Serum metabolites are represented as heat map. C, D) Pathway-specific metabolites belonging to sphingolipid (C) and fatty acid (D) metabolism are represented as heat maps. E) Model for action of ER-β in adipocytes.
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