Selective Glucocorticoid Receptor Antagonist CORT125281 Activates Brown Adipose Tissue and Alters Lipid Distribution in Male Mice

Selective Glucocorticoid Receptor Antagonist CORT125281 Activates Brown Adipose Tissue and Alters... Abstract Glucocorticoids influence a wide range of metabolic processes in the human body, and excessive glucocorticoid exposure is known to contribute to the development of metabolic disease. We evaluated the utility of the novel glucocorticoid receptor (GR) antagonist CORT125281 for its potential to overcome adiposity, glucose intolerance, and dyslipidemia and compared this head-to-head with the classic GR antagonist RU486 (mifepristone). We show that, although RU486 displays cross-reactivity to the progesterone and androgen receptor, CORT125281 selectively inhibits GR transcriptional activity. In a mouse model for diet-induced obesity, rhythmicity of circulating corticosterone levels was disturbed. CORT125281 restored this disturbed rhythmicity, in contrast to RU486, which further inhibited endogenous corticosterone levels and suppressed adrenal weight. Both CORT125281 and RU486 reduced body weight gain and fat mass. In addition, CORT125281, but not RU486, lowered plasma levels of triglycerides, cholesterol, and free fatty acids and strongly stimulated triglyceride-derived fatty acid uptake by brown adipose tissue depots. In combination with reduced lipid content in brown adipocytes, this indicates that CORT125281 enhances metabolic activity of brown adipose tissue depots. CORT125281 was also found to increase liver lipid accumulation. Taken together, CORT125281 displayed a wide range of beneficial metabolic activities that are in part distinct from RU486, but clinical utility may be limited due to liver lipid accumulation. This warrants further evaluation of GR antagonists or selective modulators that are not accompanied by liver lipid accumulation while preserving their beneficial metabolic activities. Obesity and dyslipidemia constitute major problems in modern society (1, 2), and it is increasingly being recognized that glucocorticoid (GC) stress hormones contribute to such metabolic abnormalities (3). GCs are produced in the adrenal cortex and bind to the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR), thereby regulating a wide range of processes in the human body, including lipid and glucose mobilization and disposal. Circulating GC levels display a diurnal rhythmicity, and GCs are released in response to stress. Hypothalamic-pituitary-adrenal (HPA) axis activity regulates GC secretion by a cascade of hormonal processes, initiated by release of corticotropin-releasing hormone and vasopressin by the paraventricular nucleus of the hypothalamus, which results in secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary. ACTH subsequently stimulates GC production and secretion by the adrenals. HPA axis activity is controlled by GC-mediated negative feedback on multiple levels including the inhibition of ACTH release (4, 5). Hyperactivity of the HPA axis (e.g., in Cushing syndrome) causes a myriad of metabolic adverse effects, and GR antagonists were shown to be effective in counteracting this (6). Despite being extensively used in the clinic, GR antagonist RU486 (mifepristone) lacks receptor selectivity (7) and may in certain settings also exhibit partial agonist activity (8). Therefore, the use of a selective GR antagonist that lacks partial agonistic properties may be of value. Brown adipose tissue (BAT) is a relevant metabolic target tissue of GC that has been actively pursued to combat obesity and related disorders after its discovery in humans (9). BAT effectively combusts glucose and fatty acids into heat, contributing to energy expenditure (10). BAT is activated by cold via enhanced sympathetic outflow. The norepinephrine (NE) released from sympathetic nerve terminals binds to the β3-adrenergic receptor on brown adipocytes and strongly enhances activity and expression of uncoupling protein 1 (UCP-1), the main effector protein involved in thermogenesis (11). Therapeutic targeting of BAT (e.g., with a β3-adrenergic receptor agonist) may provide an effective strategy to improve metabolic health, as it alleviates dyslipidemia, lowers blood glucose, prevents weight gain, and protects from atherosclerosis development in mice (12, 13). Accumulating evidence indicates that chronic exposure to elevated endogenous GC (14, 15) or synthetic GR agonists (16) inhibits the activity of brown adipocytes and hampers the browning of white adipose tissue (WAT) (17), although acute effects may differ between mouse and man (18). Vice versa, the classic GR antagonist RU486 was shown to acutely stimulate BAT activity (8, 15, 19). In this study, we characterized novel GR antagonist CORT125281 (20) and evaluated its effects on energy metabolism and lipid distribution in male C57BL/6J mice fed a high-fat diet (HFD). CORT125281 effectively inhibited GR activity in several cell culture models, whereas MR, progesterone receptor (PR), and androgen receptor (AR) activity was unaffected. CORT125281 inhibited weight gain and lowered plasma lipids in a model for diet-induced obesity, accompanied by robust activation of BAT in comparison with RU486. CORT125281 adversely affected hepatic lipid metabolism in mice, warranting further search for selective GR modulators that efficiently antagonize GR in BAT without adversely affecting the liver. Materials and Methods Animals All animal studies reported here have been approved by the ethical committee of Leiden University Medical Center. Mice were housed in conventional cages with a 12:12-hour light-dark cycle with ad libitum access to food and water. Ten-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were fed a control chow diet vs chow diet supplemented with CORT125281 (60 mg/kg/d) for 3 weeks (n = 8 per group); received water containing 10% fructose in combination with an HFD (60% lard, Research Diets) supplemented with vehicle, CORT125281 (60 mg/kg/d), or RU486 (also known as mifepristone; 60 mg/kg/d) for 3 weeks (n = 8 per group); or were treated with different dosages of CORT125281 by diet supplementation (6, 20, or 60 mg/kg/d) for 3 weeks (n = 7 to 8 per group). Body weight, body composition, and indirect calorimetry measurements Body weight and composition (EchoMRI-100, Houston, TX) were determined throughout all experiments. Indirect calorimetry was performed in fully automatic metabolic cages (LabMaster System; TSE Systems, Bad Homburg, Germany) from day 1 until day 6. Oxygen consumption, carbohydrate production, and caloric intake were measured and used to calculate energy expenditure and fat and carbohydrate oxidation (21). Stress-free blood collection and corticosterone measurement Stress-free blood samples (i.e., drawn within 2 minutes before plasma corticosterone levels rise) were collected after 13 days in the morning (8:30, zeitgeber time 1.5) and evening (17:30, zeitgeber time 10.5), and corticosterone levels were determined using a 125I radioimmunoassay kit (ImmuChem; MP Biochemicals, Orangeburg, NY). Plasma lipid determination After 3 weeks of treatment, blood was collected from 4-hour fasted mice to determine plasma triglycerides (TGs), plasma total cholesterol (both with enzymatic kits from Roche Diagnostics, Mannheim, Germany), and plasma free fatty acids (FFAs) (NEFA C kit; Wako Diagnostics, Instruchemie, Delfzijl, The Netherlands). Intravenous glucose tolerance test After 2 weeks, a glucose tolerance test was performed. Mice were fasted for 6 hours, and at t = 0 minutes, blood was collected. After this, mice were intravenously injected with glucose (2 g/kg), and blood was collected at t = 5, 15, 30, 60, and 120 minutes. In all samples, plasma glucose was measured using an enzymatic kit (Instruchemie). TG clearance experiment At the end of the experiment, the clearance of TGs was determined. Glycerol tri[3H]oleate ([3H]TO)–labeled lipoprotein-like emulsion particles (1.0 mg TGs in 200 µL phosphate-buffered saline) were injected intravenously in the tail vein of the mice, and blood was collected at t = 2, 5, 10, and 15 minutes (22). Mice were euthanized by cervical dislocation directly after the last blood sample and perfused with ice-cold phosphate-buffered saline for 5 minutes, and organs were harvested, weighted, and divided in pieces for messenger RNA analysis, histology, or analysis of 3H activity. Histology Metabolic organs [i.e., interscapular BAT (iBAT), gonadal WAT, and liver] were fixated in 4% paraformaldehyde for 1 day and stored in 70% ethanol until further processing. Tissues were dehydrated and embedded in paraffin, and 5-µm sections were stained for hematoxylin-eosin and Oil Red O as previously described (23). Intracellular lipid droplet size and lipid content were quantified using Image J software (version 1.47). Cell culture HEK293T cells Human HEK293T cells were transfected using Fugene HD transfection reagent (Promega, Leiden, the Netherlands) with 25 ng TAT3-luciferase (TAT3-luc), 1 ng CAGGS-renilla, 100 ng pcDNA, and 10 ng human GR, MR, AR, or PR expression vector. Cells were pretreated with different concentrations of RU486 or CORT125281 for 1 hour before exposure to 50 nM cortisol (= hydrocorticosone) (for GR, 74-fold induction of GR signaling, data not shown), 10 nM cortisol (for MR, sixfold induction of MR signaling, data not shown), 10 nM progesterone (for PR, sixfold induction of PR signaling, data not shown), or 100 nM dihydrotestosterone (DHT) (for AR, fourfold induction of AR signaling, data not shown). After 24 hours, firefly and renilla luciferase signals were measured using a Dual Luciferase assay (Promega). Cell culture murine brown adipocytes Brown preadipocytes from murine BAT depots were isolated from 5-week-old male C57BL6/J mice. Cells were reversibly immortalized by using a lentiviral vector conferring doxycylin-controlled expression of simian virus large T antigen and expanded in maintenance medium (Dulbecco’s modified Eagle medium/F12 medium supplemented with heat-inactivated fetal bovine serum, 4.5 g/L glucose, penicillin/streptomycin, and 0.1 µg/mL doxycycline). Adipogenic differentiation was induced by culturing the cells for 13 to 15 days in differentiation medium (Dulbecco’s modified Eagle medium/F12 supplemented with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, penicillin/streptomycin, 4 nM bovine insulin, 10 mM HEPES, 25 µg/mL ascorbate, and 1 µM rosiglitazone). During the last 2 days of differentiation and during the experiments, GC-free charcoal-stripped serum was used and the effects on GR transcriptional activity and BAT activity were examined in fully differentiated brown adipocytes. BAT activity was stimulated with 1 µM NE, and cells were simultaneously exposed to a combination of 10 to 1000 nM corticosterone, 10 to 1000 nM CORT125281, and/or 10 to 1000 nM RU486. After an incubation period of 8 hours, cells were lysed using TriPure (Roche, Mijdrecht, the Netherlands). RNA isolation, complementary DNA synthesis, and reverse transcription polymerase chain reaction analysis Total RNA was isolated using TriPure (Roche) according to the manufacturer’s protocol, and 500 to 1000 ng RNA was reverse-transcribed using M-MLV reverse-transcriptase (Promega). Reverse transcription polymerase chain reaction was performed on a CFX96 PCR machine using IQ SYBR-Green (Bio-Rad, Veenendaal, the Netherlands), and expression levels were normalized to housekeeping genes β2-microglobulin (B2M) or 36B4. Primer sequences are shown in Supplemental Table 1. Statistical analysis All data are presented as mean ± standard error of the mean. Statistical analyses were performed with GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA), and for mixed-model analysis, IBM SPSS 23 software was used. Statistical differences were calculated with a one-way analysis of variance with the Tukey multiple-comparison test, with a two-way analysis of variance with the Tukey multiple-comparison test, with a linear mixed model with time as the covariate, or with an unpaired t test, as appropriate. P < 0.05 was considered significant for all analyses. IC50 values were calculated using GraphPad Prism 7 software, using a nonlinear fit model. Results CORT125281 selectively inhibits GR whereas RU486 exhibits cross-reactivity for PR and AR The effect of the novel GR antagonist CORT125281 on GR transcriptional activity was examined and compared with classic GR antagonist RU486. Human HEK293T cells were transfected with GR and a TAT3-luc reporter, and this revealed the expected inhibition of cortisol-induced GR activity by both GR antagonists (20), in which RU486 was significantly more potent than CORT125281 (IC50 of 43 nM and 427 nM, respectively, P < 0.0001) (Fig. 1A). To investigate receptor selectivity, HEK293T cells were transfected with MR, PR, or AR in combination with TAT3-luc and treated with their respective agonists around their estimated EC90 concentration (24). Neither CORT125281 nor RU486 affected cortisol-induced MR signaling (Fig. 1B). Although RU486 potently inhibited PR signaling (IC50: 0.6 nM) (Fig. 1C) and also displayed moderate inhibitory actions on AR signaling (IC50: 4.1 µM) (Fig. 1D), CORT125281 did not affect progesterone-induced PR signaling and DHT-induced AR signaling (Fig. 1C and 1D). Taken together, this supports the notion that CORT125281 is a selective GR antagonist, whereas RU486 exhibits cross-reactivity for PR and AR. Figure 1. View largeDownload slide The effect of GR antagonists on nuclear receptor signaling in vitro. HEK293T cells transfected with a TAT3-luc reporter were used to determine the antagonistic effects of CORT125281 and RU486 on (A) corticosterone-induced (10 nM) GR signaling, (B) corticosterone-induced (50 nM) MR signaling, (C) progesterone-induced (10 nM) PR signaling, and (D) DHT-induced (100 nM) AR signaling. Statistical significance was calculated using two-way analysis of variance with the Bonferroni multiple-comparisons test. **P < 0.01 vs CORT125281, ****P < 0.0001 vs CORT125281. Figure 1. View largeDownload slide The effect of GR antagonists on nuclear receptor signaling in vitro. HEK293T cells transfected with a TAT3-luc reporter were used to determine the antagonistic effects of CORT125281 and RU486 on (A) corticosterone-induced (10 nM) GR signaling, (B) corticosterone-induced (50 nM) MR signaling, (C) progesterone-induced (10 nM) PR signaling, and (D) DHT-induced (100 nM) AR signaling. Statistical significance was calculated using two-way analysis of variance with the Bonferroni multiple-comparisons test. **P < 0.01 vs CORT125281, ****P < 0.0001 vs CORT125281. CORT125281 reverses corticosterone-mediated GR activity in murine brown adipocytes in vitro To assess whether CORT125281 influences the activity of brown adipocytes, we used cell lines derived from murine BAT depots. Preadipocytes were differentiated into mature brown adipocytes and treated with corticosterone in combination with RU486 or CORT125281. To determine the effect of both compounds on GR transcriptional activity, we measured the expression of the well-known GR target genes Fkbp5 and Gilz (25, 26). Murine brown adipocytes were responsive to corticosterone, as treatment with 10 nM corticosterone significantly upregulated expression of Fkbp5 and Gilz (Fig. 2A and 2B). Treatment with either RU486 or CORT125281 effectively inhibited corticosterone-induced GR transcriptional activity (Fig. 2A and 2B). Also, for GR transcriptional activity induced by 1 µM corticosterone, both GR antagonists significantly inhibited GR target gene expression, although GR inhibition by 1 µM RU486 was stronger compared with 1 µM CORT125281, likely reflecting differences in binding affinity (Supplemental Fig. 1A and 1B) (20). As expected, activity of murine brown adipocytes was inhibited by corticosterone, that is, decreased NE-induced UCP-1 expression upon 10 nM (Fig. 2C) and 1 µM corticosterone exposure (Supplemental Fig. 1C). Both CORT125281 and RU486 were able to (partially) prevent corticosterone-induced inhibition of BAT activity, as coincubation with the GR antagonists results in enhanced UCP-1 expression (Fig. 2C, Supplemental Fig. 1C). Of note, RU486 did not dose-dependently reverse corticosterone-inhibited UCP-1 expression (Fig. 2C) and was not able, even at high doses, to fully prevent corticosterone-inhibited UCP-1 expression (Supplemental Fig. 1C). These findings may be explained by partial agonistic properties of RU486 on the GR. To test this, mature brown adipocytes were treated with different doses of RU486 or CORT125281, and this was compared with the agonistic effect of 10 nM corticosterone. Treatment with RU486 resulted in upregulation of the GR target gene Fkbp5 (P < 0.05) but not Gilz, whereas CORT125281 did not influence Fkbp5 or Gilz expression (Fig. 2D). RU486 treatment tended to reduce NE-induced Ucp1 expression (−40% vs vehicle), which is significant compared with 10 nM CORT125281 (P < 0.01, Fig. 2D). Taken together, these data suggest partial agonistic properties of RU486, which could limit BAT activating capacity by RU486, whereas CORT125281 showed only antagonistic properties on brown adipocytes. Figure 2. View largeDownload slide The effects of GR antagonists on murine BAT cells in vitro. RU486 and CORT125281 antagonistic properties on 10 nM corticosterone-regulated gene expression (A) Fkbp5 and (B) Gilz. **P < 0.01 vs NE, ***P < 0.001 vs NE, ****P < 0.0001 vs NE, $P < 0.05 vs CORT + NE, $$P < 0.01 vs CORT + NE, $$$$P < 0.0001 vs CORT + NE. (C) The effect of CORT125281 and RU486 on Ucp1 expression in murine brown adipocytes after 8 hours of exposure. ***P < 0.001, ****P < 0.0001, $$$P < 0.001 vs CORT, $$$$P < 0.0001 vs CORT, ^P < 0.05 vs CORT + NE, ^^P < 0.01 vs CORT + NE. (D) Assessment of partial agonism by RU486 and CORT125281 compared with 10 nM corticosterone on the expression of Fkbp5, Gilz, and UCP1 in murine brown adipocytes after 8 hours of exposure. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. CORT, corticosterone; Veh, vehicle. Figure 2. View largeDownload slide The effects of GR antagonists on murine BAT cells in vitro. RU486 and CORT125281 antagonistic properties on 10 nM corticosterone-regulated gene expression (A) Fkbp5 and (B) Gilz. **P < 0.01 vs NE, ***P < 0.001 vs NE, ****P < 0.0001 vs NE, $P < 0.05 vs CORT + NE, $$P < 0.01 vs CORT + NE, $$$$P < 0.0001 vs CORT + NE. (C) The effect of CORT125281 and RU486 on Ucp1 expression in murine brown adipocytes after 8 hours of exposure. ***P < 0.001, ****P < 0.0001, $$$P < 0.001 vs CORT, $$$$P < 0.0001 vs CORT, ^P < 0.05 vs CORT + NE, ^^P < 0.01 vs CORT + NE. (D) Assessment of partial agonism by RU486 and CORT125281 compared with 10 nM corticosterone on the expression of Fkbp5, Gilz, and UCP1 in murine brown adipocytes after 8 hours of exposure. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. CORT, corticosterone; Veh, vehicle. CORT125281 reduces body weight, fat mass, and plasma lipids in HFD-fed mice To evaluate the effects of CORT125281 on metabolism in a whole organism, a mouse model for diet-induced obesity was used. HFD-fed mice were treated with either CORT125281 (60 mg/kg/d) or the classic GR antagonist RU486 (60 mg/kg/d). For comparison, we also performed an experiment in which chow-fed mice were treated by diet supplementation with CORT125281 (60 mg/kg/d). At equal dosage, CORT125281 and RU486 similarly reduced HFD-induced body weight gain with ~10% (P < 0.001 for CORT125281 at day 21, Fig. 3A), whereas body weight was not altered in chow-fed mice (Supplemental Fig. 2A). Both GR antagonists significantly reduced fat mass but not lean mass in HFD-fed mice (−23% for RU486 and −32% for CORT125281 at day 21, P < 0.01 and P < 0.0001, respectively) (Fig. 3B and 3C), whereas CORT125281 did not affect fat mass or lean mass in chow-fed mice (Supplemental Fig. 2B and 2C). In the HFD condition, treatment with CORT125281 significantly lowered plasma TGs (−56%, P < 0.0001) (Fig. 3D) and cholesterol levels (−30%, P < 0.05) (Fig. 3E) compared with vehicle and RU486-treated mice, as well as significantly lowered plasma FFAs compared with RU486-treated mice (−23%, P < 0.05) (Fig. 3F). Similar plasma lipid-lowering activities of CORT125281 were found in chow-fed mice (i.e., significant reduction of plasma TGs and cholesterol and a near-significant reduction of FFAs) (Supplemental Fig. 2D–2F). In a subsequent experiment, different dosages of CORT125281 (6, 20, or 60 mg/kg/d) were evaluated in HFD-fed mice, which revealed that CORT125281 seemed to reduce body weight, fat mass, plasma TGs, cholesterol, and FFAs in a dose-dependent manner, with no effect on lean mass (Supplemental Fig. 3A–3F). In addition to lipid metabolism, we investigated the effects of CORT125281 on glucose metabolism. This revealed that CORT125281 did not affect basal glucose levels or intravenous glucose tolerance (Fig. 3G and 3H), whereas RU486 significantly improved glucose tolerance in HFD-fed mice, to a similar degree as previously described (27). Altogether, these data suggest that CORT125281 treatment reduces diet-induced weight gain and body fat mass and that CORT125281 effectively lowers plasma lipids. Figure 3. View largeDownload slide The effect of GR antagonists on body weight, body composition, and plasma lipids and glucose of HFD-fed C57BL/6J mice. The effect of the classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) body weight, (B) body fat mass, and (C) lean mass. The effect on (D) plasma TGs, (E) plasma total cholesterol (TC), and (F) plasma FFAs after 3 weeks of treatment. The effect on (G) basal glucose levels and (H) intravenous glucose tolerance (ivGTT) after 2 weeks of treatment. Statistical significance was calculated using (A–C) a mixed-model analysis, (D–G) a one-way analysis of variance with the Tukey multiple-comparisons test, or (H) a two-way analysis of variance with the Tukey multiple-comparisons test. ~P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 3. View largeDownload slide The effect of GR antagonists on body weight, body composition, and plasma lipids and glucose of HFD-fed C57BL/6J mice. The effect of the classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) body weight, (B) body fat mass, and (C) lean mass. The effect on (D) plasma TGs, (E) plasma total cholesterol (TC), and (F) plasma FFAs after 3 weeks of treatment. The effect on (G) basal glucose levels and (H) intravenous glucose tolerance (ivGTT) after 2 weeks of treatment. Statistical significance was calculated using (A–C) a mixed-model analysis, (D–G) a one-way analysis of variance with the Tukey multiple-comparisons test, or (H) a two-way analysis of variance with the Tukey multiple-comparisons test. ~P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CORT125281 restores HFD-disturbed HPA axis activity Under chow-fed conditions, mice display a circadian rhythm in circulating corticosterone with peak levels before the dark phase (i.e., start of the active period, data not shown). Three weeks of HFD resulted in dampening of the circadian corticosterone rhythm, as morning and evening circulating corticosterone levels were similar (Fig. 4A). RU486 treatment resulted in lower circulating corticosterone levels in the evening, suggesting an agonistic effect on the HPA axis resulting in enhanced negative feedback under this dose regimen (−80%, P < 0.01) (Fig. 4A). Strikingly, CORT125281 restored the HFD-disturbed circadian corticosterone rhythm (P < 0.0001, morning vs evening), and substantially higher circulating corticosterone levels in the evening were found compared with vehicle HFD-fed mice (P < 0.0001) (Fig. 4A). The lack of agonistic activity of CORT125281 is further supported by the observation that CORT125281 did not influence adrenal weight, whereas RU486 induced adrenal atrophy (−51% organ weight, P < 0.05) (Fig. 4B, Supplemental Fig. 4A), indicating continuous negative feedback on the HPA axis. At all evaluated dosages, CORT125281 did not influence the weight of the thymus and spleen (Fig. 4C and 4D, Supplemental Fig. 4B and 4C), organs that involute after chronic GC exposure, whereas RU486 was found to reduce thymus weight (−47%, P < 0.05) (Fig. 4C). Figure 4. View largeDownload slide The effect of GR antagonists on endogenous corticosterone and GC-sensitive organ weight in HFD-fed C57BL/6J mice. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) circulating corticosterone levels in the morning and evening and on (B) adrenal, (C) thymus, and (D) spleen weight. Statistical significance was calculated using a two-way analysis of variance with the (A) Tukey multiple-comparisons test or (B–D) a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, ****P < 0.0001, $$P < 0.01 vs vehicle in the evening, $$$$P < 0.0001 vs vehicle in the evening. AM, morning; NS, not significant; PM, evening; Veh, vehicle. Figure 4. View largeDownload slide The effect of GR antagonists on endogenous corticosterone and GC-sensitive organ weight in HFD-fed C57BL/6J mice. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) circulating corticosterone levels in the morning and evening and on (B) adrenal, (C) thymus, and (D) spleen weight. Statistical significance was calculated using a two-way analysis of variance with the (A) Tukey multiple-comparisons test or (B–D) a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, ****P < 0.0001, $$P < 0.01 vs vehicle in the evening, $$$$P < 0.0001 vs vehicle in the evening. AM, morning; NS, not significant; PM, evening; Veh, vehicle. CORT125281 stimulates fatty acid uptake and combustion by interscapular BAT To monitor energy expenditure, mice were housed in fully automated metabolic cages during the first week of treatment. RU486 significantly increased total energy expenditure, whereas CORT125281 did not (Fig. 5A). Treatment with RU486 and CORT125281 both resulted in increased fat oxidation (Fig. 5B) and decreased carbohydrate oxidation (Fig. 5C), as evident from a lowered respiratory exchange ratio (Fig. 5D). We next investigated the fate of intravenously injected lipoprotein-like particles labeled with [3H]TO lipids. In mice treated with CORT125281 but not RU486, plasma decay of [3H]TO was more rapid (P < 0.01, Fig. 5E), indicating enhanced TG uptake from plasma. Uptake of [3H]TO-derived activity by iBAT and dorso-cervical BAT was significantly increased in the CORT125281 group (+115%, P < 0.0001 and +61%, P < 0.05, respectively) (Fig. 5F), indicating enhanced metabolic activity of these BAT depots. This is further supported by a tendency toward reduced iBAT weight (Fig. 5G, Supplemental Fig. 5A), accompanied by reduced lipid content in CORT125281-treated mice compared with RU486-treated mice (−51%, P < 0.05) (Fig. 5H and 5I). In addition, CORT125281- and RU486-treated mice showed decreased gonadal WAT weight and smaller average cell size (Fig. 5J–5L, Supplemental Fig. 5B). Collectively, these data suggest that CORT125281 activated BAT to stimulate fatty acid uptake and combustion, whereas RU486 did not. Figure 5. View largeDownload slide The effect of GR antagonists on the activity of metabolic organs. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) energy expenditure (EE), (B) fat oxidation, (C) carbohydrate oxidation, and (D) respiratory exchange ratio. The effect on (E) plasma decay and (F) uptake of lipoprotein TG-derived FFA by metabolic tissues. The effect on (G) iBAT weight and (H) lipid content, as well as (I) representative images of hematoxylin and eosin–stained iBAT slices. The effect on (J) gWAT weight and (K) average cell size, as well as (L) representative images of hematoxylin and eosin–stained gWAT slices. Statistical significance was calculated using a (A–D, G, H, J, K) one-way analysis of variance with the Tukey multiple-comparisons test or (E, F) two-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$P < 0.01 vs RU486, $$$$P < 0.0001 vs RU486. dcBAT, dorsocervical BAT; gWAT, gonadal WAT; sWAT, subcutaneous white adipose tissue; Veh, vehicle; vWAT, visceral white adipose tissue. Figure 5. View largeDownload slide The effect of GR antagonists on the activity of metabolic organs. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) energy expenditure (EE), (B) fat oxidation, (C) carbohydrate oxidation, and (D) respiratory exchange ratio. The effect on (E) plasma decay and (F) uptake of lipoprotein TG-derived FFA by metabolic tissues. The effect on (G) iBAT weight and (H) lipid content, as well as (I) representative images of hematoxylin and eosin–stained iBAT slices. The effect on (J) gWAT weight and (K) average cell size, as well as (L) representative images of hematoxylin and eosin–stained gWAT slices. Statistical significance was calculated using a (A–D, G, H, J, K) one-way analysis of variance with the Tukey multiple-comparisons test or (E, F) two-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$P < 0.01 vs RU486, $$$$P < 0.0001 vs RU486. dcBAT, dorsocervical BAT; gWAT, gonadal WAT; sWAT, subcutaneous white adipose tissue; Veh, vehicle; vWAT, visceral white adipose tissue. CORT125281 increases liver lipid content As GR activity is known to influence hepatic function (e.g., lipid uptake, very-low-density lipoprotein production, de novo lipogenesis) (14), we next analyzed the livers of mice treated with RU486 and CORT125281. In HFD-fed mice, CORT125281 significantly increased liver weight (+51%, P < 0.01) (Fig. 6A, Supplemental Fig. 5C) and treatment with CORT125281 was accompanied by increased liver TGs (+88%, P < 0.05) (Supplemental Fig. 5D) and total liver lipids (visualized by an Oil Red O staining, Fig. 6B and 6C). Also, in chow-fed mice, CORT125281 seemed to increase liver weight and significantly increased liver TGs (Supplemental Fig. 2G and 2H). Of note, both RU486 and CORT125281 strongly upregulated expression of Cyp3a11, suggestive of induction of the pregnane X receptor (PXR) (Fig. 6D). PXR agonism is known to cause hepatic lipid accumulation, at least partly via increased hepatic expression of fatty acid transporter Cd36 (28), which we also observed for both CORT125281 and RU486 (Fig. 6E). Although RU486 induced Cyp3a11 expression most strongly, this was not accompanied by the highest hepatic lipid content (Fig. 6B–6E), suggesting additional, differential effects of RU486 and CORT125281 on liver lipid metabolism. Figure 6. View largeDownload slide The effect of GR antagonists on the liver. The effect of the novel GR antagonist CORT125281 and the classic GR antagonist RU486 on (A) liver weight and (B) liver Oil Red O. (C) Representative images of Oil Red O–stained liver slices. Hepatic expression of (D) Cyp3a11 and (E) Cd36. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. **P < 0.01 vs Veh, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$$$P < 0.0001 vs RU486. Veh, vehicle. Figure 6. View largeDownload slide The effect of GR antagonists on the liver. The effect of the novel GR antagonist CORT125281 and the classic GR antagonist RU486 on (A) liver weight and (B) liver Oil Red O. (C) Representative images of Oil Red O–stained liver slices. Hepatic expression of (D) Cyp3a11 and (E) Cd36. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. **P < 0.01 vs Veh, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$$$P < 0.0001 vs RU486. Veh, vehicle. Discussion In this study, we describe the effects of a novel GR antagonist CORT125281 on metabolism and HPA axis activity in a model for diet-induced obesity in male mice. In our studies, we compared CORT125281 head-to-head with the classic GR antagonist RU486. First, we characterized the GR specificity of CORT125281, and luciferase reporter experiments clearly show that CORT125281 selectively inhibits the GR, whereas the classic GR antagonist RU486 also inhibits PR and AR transcriptional activity, as expected (7, 29). In a model for diet-induced obesity, we have shown that CORT125281 and RU486 equally reduce body weight gain in HFD-fed mice, similar to previous observations for RU486 (30). In addition, both GR antagonists reduce total fat mass without adversely affecting the lean mass. CORT125281, but not RU486, significantly lowered plasma lipids, restored circadian corticosterone rhythmicity, and induced fatty acid uptake and combustion by iBAT. Both GR antagonists effectively reversed corticosterone-suppressed UCP-1 expression in brown adipocytes in vitro, and this reversal of corticosterone-inhibitory actions on BAT could partially underlie the CORT125281-induced BAT activity observed in vivo. Although RU486 may activate BAT in specific contexts (8), in the present setting, RU486 did not seem to activate BAT in vivo, and this discrepancy between RU486 and CORT125281 may be explained by differential effects on PR or AR activity. Alternatively, because ACTH was shown to stimulate BAT activity (15), the restored HPA axis activity in CORT125281-treated mice may augment circulating ACTH levels and thereby enhance ACTH-induced BAT activation. The lack of partial agonism on the HPA axis by CORT125281 may thus underlie the differential effects of RU486 and CORT125281 on BAT activity in vivo. Additional partial agonistic activities of RU486 (as evident from reduced NE-induced UCP-1 expression in RU486-treated brown adipocytes in vitro, Fig. 2D) may also explain the lower BAT-activating capacity of RU486. In our study, we observed disturbed corticosterone rhythmicity upon HFD, which is in line with a previous study (31). Nutrient sensors influence the peripheral clock (32), and HFD was shown to alter diurnal patterns of leptin and insulin, as well as to reduce circadian patterns of clock genes in metabolic tissues (33). Thus, the flattened corticosterone rhythm observed in our study could be a consequence of HFD-disturbed circadian rhythm, which is supported by the observation that the HFD-fed mice in our study eat throughout the whole day rather than mainly in the dark period (data not shown). Alternatively, HFD could influence the HPA axis and its hormones directly, as decreased 11β-HSD1 expression (which converts inactive into active GC) and altered corticotropin-releasing hormone and GR expression in the paraventricular nucleus were observed upon HFD feeding (31). Fatty acids were also shown to regulate circulating corticosterone levels, and fatty acid sensors are known to be present in the hypothalamus (34). FFA-lowering strategies (e.g., insulin administration) were shown to increase plasma ACTH and corticosterone levels (35), and based on this, the decreased plasma FFA levels upon CORT125281 treatment could contribute to the restored corticosterone rhythmicity observed in our study. In addition, the lack of peripheral negative GC feedback on the HPA axis, due to the continuous presence of CORT125281, could contribute to the restored corticosterone rhythmicity. Although acute RU486 treatment can interfere with GR-mediated negative feedback and disinhibit the HPA axis (36), in the present setting (continuous administration of high dose via the food), both corticosterone levels and adrenal weights were strongly reduced, suggesting suppression of ACTH release rather than classic disinhibition. Although the reduced plasma lipids upon CORT125281 treatment can partially be attributed to enhanced BAT activity, it seems likely that enhanced lipid uptake by the liver is also involved. HFD induces hepatic expression of the cellular fatty acid transporter CD36 (37). CD36 mediates hepatic lipid uptake and is critically involved in the pathogenesis of liver steatosis as its upregulation induces lipid accumulation in the liver (38), and hepatic deletion of CD36 prevents this (39). Both endogenous GC (14, 40) and synthetic GC agonists (41, 42) have been shown to increase hepatic CD36 expression, thereby aggravating liver steatosis (14). Vice versa, GR knockout decreases hepatic CD36 expression and subsequently lowers liver lipids (43). Surprisingly, treatment with CORT125281 and RU486 also increased hepatic Cd36 expression, which is likely attributed to activation of the xenobiotic sensor PXR (44). This subsequently enhances fatty acid uptake, resulting in lipid accumulation and enhanced liver weight in mice. Currently, it is unknown if CORT125281 induces similar hepatic lipid accumulation in humans. Remarkably, in our studies, RU486 treatment is not associated with lipid accumulation, whereas RU486 is a known PXR ligand (28) and enhanced hepatic Cd36 and Cyp3a11 expression. This suggests additional lipid-lowering activities of RU486 (e.g., very-low-density lipoprotein production, β-oxidation) that prevent hepatic lipid uptake and accumulation and the development of steatosis. The differential effects of RU486 and CORT125281 may therefore be a consequence of the partial agonistic features of RU486 that are lacking in CORT125281. To date, the utility of GR antagonists could be further improved for the treatment of metabolic disease. Based on our current study, GR antagonism with RU486 affects only body weight and fat mass but does not display additional beneficial metabolic activities, whereas the potential of CORT125281 may be limited due to adverse liver steatosis–inducing effects that we observe in mice. This may call for GR ligands that selectively act on BAT or that exhibit mixed agonistic and antagonistic features (45–48), to exploit the beneficial metabolic effects of both GR agonism and GR antagonism. Abbreviations: ACTH adrenocorticotropic hormone AR androgen receptor BAT brown adipose tissue DHT dihydrotestosterone FFA free fatty acid GC glucocorticoid GR glucocorticoid receptor HFD high-fat diet HPA hypothalamic-pituitary-adrenal iBAT interscapular brown adipose tissue MR mineralocorticoid receptor NE norepinephrine PR progesterone receptor PXR pregnane X receptor TAT3-luc TAT3-luciferase TG triglyceride UCP-1 uncoupling protein 1 WAT white adipose tissue. Acknowledgments We thank Trea Streefland for valuable technical assistance; Jia Liu and Antoine de Vries from Leiden University Medical Center’s Laboratory of Experimental Cardiology, as well as Sander Kooijman from the Department of Medicine, for generating lines of reversibly immortalized murine brown preadipocytes; and Dr. A. O. Brinkmann for providing the human PR and AR constructs. Financial Support: This study was partially funded by Corcept Therapeutics. L.L.K. was funded with a grant by the Board of Directors of Leiden University Medical Center. Disclosure Summary: Hazel Hunt is an employee of Corcept Therapeutics, a company that develops selective receptor modulators, including CORT125281. The remaining authors have nothing to disclose. References 1. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. JAMA . 2007; 298( 3): 299– 308. Google Scholar CrossRef Search ADS PubMed  2. Phillips CM, Tierney AC, Perez-Martinez P, Defoort C, Blaak EE, Gjelstad IM, Lopez-Miranda J, Kiec-Klimczak M, Malczewska-Malec M, Drevon CA, Hall W, Lovegrove JA, Karlstrom B, Risérus U, Roche HM. Obesity and body fat classification in the metabolic syndrome: impact on cardiometabolic risk metabotype. Obesity (Silver Spring) . 2013; 21( 1): E154– E161. Google Scholar CrossRef Search ADS PubMed  3. Walker BR. Cortisol—cause and cure for metabolic syndrome? Diabet Med . 2006; 23( 12): 1281– 1288. Google Scholar CrossRef Search ADS PubMed  4. Shipston MJ. Mechanism(s) of early glucocorticoid inhibition of adrenocorticotropin secretion from anterior pituitary corticotropes. Trends Endocrinol Metab . 1995; 6( 8): 261– 266. Google Scholar CrossRef Search ADS PubMed  5. Herman JP, McKlveen JM, Solomon MB, Carvalho-Netto E, Myers B. Neural regulation of the stress response: glucocorticoid feedback mechanisms. Braz J Med Biol Res . 2012; 45( 4): 292– 298. Google Scholar CrossRef Search ADS PubMed  6. Castinetti F, Brue T, Conte-Devolx B. The use of the glucocorticoid receptor antagonist mifepristone in Cushing’s syndrome. Curr Opin Endocrinol Diabetes Obes . 2012; 19( 4): 295– 299. Google Scholar CrossRef Search ADS PubMed  7. Gaillard RC, Riondel A, Muller AF, Herrmann W, Baulieu EE. RU 486: a steroid with antiglucocorticosteroid activity that only disinhibits the human pituitary-adrenal system at a specific time of day. Proc Natl Acad Sci USA . 1984; 81( 12): 3879– 3882. Google Scholar CrossRef Search ADS PubMed  8. van den Heuvel JK, Boon MR, van Hengel I, Peschier-van der Put E, van Beek L, van Harmelen V, van Dijk KW, Pereira AM, Hunt H, Belanoff JK, Rensen PC, Meijer OC. Identification of a selective glucocorticoid receptor modulator that prevents both diet-induced obesity and inflammation. Br J Pharmacol . 2016; 173( 11): 1793– 1804. Google Scholar CrossRef Search ADS PubMed  9. Poekes L, Lanthier N, Leclercq IA. Brown adipose tissue: a potential target in the fight against obesity and the metabolic syndrome. Clin Sci (Lond) . 2015; 129( 11): 933– 949. Google Scholar CrossRef Search ADS PubMed  10. Kooijman S, van den Heuvel JK, Rensen PC. Neuronal control of brown fat activity. Trends Endocrinol Metab . 2015; 26( 11): 657– 668. Google Scholar CrossRef Search ADS PubMed  11. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev . 2004; 84( 1): 277– 359. Google Scholar CrossRef Search ADS PubMed  12. Warner A, Kjellstedt A, Carreras A, Böttcher G, Peng XR, Seale P, Oakes N, Lindén D. Activation of β3-adrenoceptors increases in vivo free fatty acid uptake and utilization in brown but not white fat depots in high-fat-fed rats. Am J Physiol Endocrinol Metab . 2016; 311( 6): E901– E910. Google Scholar CrossRef Search ADS PubMed  13. Berbée JF, Boon MR, Khedoe PP, Bartelt A, Schlein C, Worthmann A, Kooijman S, Hoeke G, Mol IM, John C, Jung C, Vazirpanah N, Brouwers LP, Gordts PL, Esko JD, Hiemstra PS, Havekes LM, Scheja L, Heeren J, Rensen PC. Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development. Nat Commun . 2015; 6: 6356. Google Scholar CrossRef Search ADS PubMed  14. van den Beukel JC, Boon MR, Steenbergen J, Rensen PC, Meijer OC, Themmen AP, Grefhorst A. Cold exposure partially corrects disturbances in lipid metabolism in a male mouse model of glucocorticoid excess. Endocrinology . 2015; 156( 11): 4115– 4128. Google Scholar CrossRef Search ADS PubMed  15. van den Beukel JC, Grefhorst A, Quarta C, Steenbergen J, Mastroberardino PG, Lombès M, Delhanty PJ, Mazza R, Pagotto U, van der Lely AJ, Themmen AP. Direct activating effects of adrenocorticotropic hormone (ACTH) on brown adipose tissue are attenuated by corticosterone. FASEB J . 2014; 28( 11): 4857– 4867. Google Scholar CrossRef Search ADS PubMed  16. Soumano K, Desbiens S, Rabelo R, Bakopanos E, Camirand A, Silva JE. Glucocorticoids inhibit the transcriptional response of the uncoupling protein-1 gene to adrenergic stimulation in a brown adipose cell line. Mol Cell Endocrinol . 2000; 165( 1–2): 7– 15. Google Scholar CrossRef Search ADS PubMed  17. Kong X, Yu J, Bi J, Qi H, Di W, Wu L, Wang L, Zha J, Lv S, Zhang F, Li Y, Hu F, Liu F, Zhou H, Liu J, Ding G. Glucocorticoids transcriptionally regulate miR-27b expression promoting body fat accumulation via suppressing the browning of white adipose tissue. Diabetes . 2014; 64( 2): 393– 404. Google Scholar CrossRef Search ADS PubMed  18. Ramage LE, Akyol M, Fletcher AM, Forsythe J, Nixon M, Carter RN, van Beek EJ, Morton NM, Walker BR, Stimson RH. Glucocorticoids acutely increase brown adipose tissue activity in humans, revealing species-specific differences in UCP-1 regulation. Cell Metab . 2016; 24( 1): 130– 141. Google Scholar CrossRef Search ADS PubMed  19. Rodríguez AM, Palou A. The steroid RU486 induces UCP1 expression in brown adipocytes. Pflugers Arch . 2004; 449( 2): 170– 174. Google Scholar CrossRef Search ADS PubMed  20. Hunt HJ, Belanoff JK, Walters I, Gourdet B, Thomas J, Barton N, Unitt J, Phillips T, Swift D, Eaton E. Identification of the clinical candidate (R)-(1-(4-Fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone (CORT125134): a selective glucocorticoid receptor (GR) antagonist. J Med Chem . 2017; 60( 8): 3405– 3421. Google Scholar CrossRef Search ADS PubMed  21. Van Klinken JB, van den Berg SA, Havekes LM, Willems Van Dijk K. Estimation of activity related energy expenditure and resting metabolic rate in freely moving mice from indirect calorimetry data. PLoS One . 2012; 7( 5): e36162. Google Scholar CrossRef Search ADS PubMed  22. Rensen PC, Herijgers N, Netscher MH, Meskers SC, van Eck M, van Berkel TJ. Particle size determines the specificity of apolipoprotein E–containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo. J Lipid Res . 1997; 38( 6): 1070– 1084. Google Scholar PubMed  23. Kooijman S, Boon MR, Parlevliet ET, Geerling JJ, van de Pol V, Romijn JA, Havekes LM, Meurs I, Rensen PC. Inhibition of the central melanocortin system decreases brown adipose tissue activity. J Lipid Res . 2014; 55( 10): 2022– 2032. Google Scholar CrossRef Search ADS PubMed  24. Kizu R, Otsuki N, Kishida Y, Toriba A, Mizokami A, Burnstein KL, Klinge CM, Hayakawai K. A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells. Anal Sci . 2004; 20( 1): 55– 59. Google Scholar CrossRef Search ADS PubMed  25. Zalachoras I, Verhoeve SL, Toonen LJ, van Weert LT, van Vlodrop AM, Mol IM, Meelis W, de Kloet ER, Meijer OC. Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression. Mol Psychiatry . 2016; 21( 12): 1733– 1739. Google Scholar CrossRef Search ADS PubMed  26. Drebert Z, Bracke M, Beck IM. Glucocorticoids and the non-steroidal selective glucocorticoid receptor modulator, compound A, differentially affect colon cancer–derived myofibroblasts. J Steroid Biochem Mol Biol . 2015; 149: 92– 105. Google Scholar CrossRef Search ADS PubMed  27. Mammi C, Marzolla V, Armani A, Feraco A, Antelmi A, Maslak E, Chlopicki S, Cinti F, Hunt H, Fabbri A, Caprio M. A novel combined glucocorticoid-mineralocorticoid receptor selective modulator markedly prevents weight gain and fat mass expansion in mice fed a high-fat diet. Int J Obes . 2016; 40( 6): 964– 972. Google Scholar CrossRef Search ADS   28. Zhou C, King N, Chen KY, Breslow JL. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice. J Lipid Res . 2009; 50( 10): 2004– 2013. Google Scholar CrossRef Search ADS PubMed  29. Schreiber JR, Hsueh AJ, Baulieu EE. Binding of the anti-progestin RU-486 to rat ovary steroid receptors. Contraception . 1983; 28( 1): 77– 85. Google Scholar CrossRef Search ADS PubMed  30. Asagami T, Belanoff JK, Azuma J, Blasey CM, Clark RD, Tsao PS. Selective glucocorticoid receptor (GR-II) antagonist reduces body weight gain in mice. J Nutr Metab . 2011; 2011: 235389. Google Scholar CrossRef Search ADS PubMed  31. Auvinen HE, Romijn JA, Biermasz NR, Pijl H, Havekes LM, Smit JW, Rensen PC, Pereira AM. The effects of high fat diet on the basal activity of the hypothalamus-pituitary-adrenal axis in mice. J Endocrinol . 2012; 214( 2): 191– 197. Google Scholar CrossRef Search ADS PubMed  32. Oosterman JE, Kalsbeek A, la Fleur SE, Belsham DD. Impact of nutrients on circadian rhythmicity. Am J Physiol Regul Integr Comp Physiol . 2014; 308( 5): R337– R350. Google Scholar CrossRef Search ADS PubMed  33. Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y, Turek FW, Bass J. High-fat diet disrupts behavioral and molecular circadian rhythms in mice. Cell Metab . 2007; 6( 5): 414– 421. Google Scholar CrossRef Search ADS PubMed  34. Lam TK, Schwartz GJ, Rossetti L. Hypothalamic sensing of fatty acids. Nat Neurosci . 2005; 8( 5): 579– 584. Google Scholar CrossRef Search ADS PubMed  35. Oh YT, Oh KS, Kang I, Youn JH. A fall in plasma free fatty acid (FFA) level activates the hypothalamic-pituitary-adrenal axis independent of plasma glucose: evidence for brain sensing of circulating FFA. Endocrinology . 2012; 153( 8): 3587– 3592. Google Scholar CrossRef Search ADS PubMed  36. Ratka A, Sutanto W, Bloemers M, de Kloet ER. On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation. Neuroendocrinology . 1989; 50( 2): 117– 123. Google Scholar CrossRef Search ADS PubMed  37. Sheedfar F, Sung MM, Aparicio-Vergara M, Kloosterhuis NJ, Miquilena-Colina ME, Vargas-Castrillón J, Febbraio M, Jacobs RL, de Bruin A, Vinciguerra M, García-Monzón C, Hofker MH, Dyck JR, Koonen DP. Increased hepatic CD36 expression with age is associated with enhanced susceptibility to nonalcoholic fatty liver disease. Aging (Albany NY) . 2014; 6( 4): 281– 295. Google Scholar CrossRef Search ADS PubMed  38. Yao L, Wang C, Zhang X, Peng L, Liu W, Zhang X, Liu Y, He J, Jiang C, Ai D, Zhu Y. Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice. Hepatology . 2016; 64( 1): 92– 105. Google Scholar CrossRef Search ADS PubMed  39. Wilson CG, Tran JL, Erion DM, Vera NB, Febbraio M, Weiss EJ. Hepatocyte-specific disruption of CD36 attenuates fatty liver and improves insulin sensitivity in HFD-fed mice. Endocrinology . 2016; 157( 2): 570– 585. Google Scholar CrossRef Search ADS PubMed  40. Bowles NP, Karatsoreos IN, Li X, Vemuri VK, Wood JA, Li Z, Tamashiro KL, Schwartz GJ, Makriyannis AM, Kunos G, Hillard CJ, McEwen BS, Hill MN. A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome. Proc Natl Acad Sci USA . 2014; 112( 1): 285– 290. Google Scholar CrossRef Search ADS PubMed  41. Poggioli R, Ueta CB, Drigo RA, Castillo M, Fonseca TL, Bianco AC. Dexamethasone reduces energy expenditure and increases susceptibility to diet-induced obesity in mice. Obesity (Silver Spring) . 2013; 21( 9): E415– E420. Google Scholar PubMed  42. Feng B, He Q, Xu H. FOXO1-dependent up-regulation of MAP kinase phosphatase 3 (MKP-3) mediates glucocorticoid-induced hepatic lipid accumulation in mice. Mol Cell Endocrinol . 2014; 393( 1–2): 46– 55. Google Scholar CrossRef Search ADS PubMed  43. Lemke U, Krones-Herzig A, Berriel Diaz M, Narvekar P, Ziegler A, Vegiopoulos A, Cato AC, Bohl S, Klingmüller U, Screaton RA, Müller-Decker K, Kersten S, Herzig S. The glucocorticoid receptor controls hepatic dyslipidemia through Hes1. Cell Metab . 2008; 8( 3): 212– 223. Google Scholar CrossRef Search ADS PubMed  44. Bitter A, Rümmele P, Klein K, Kandel BA, Rieger JK, Nüssler AK, Zanger UM, Trauner M, Schwab M, Burk O. Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Arch Toxicol . 2014; 89( 11): 2089– 2103. Google Scholar CrossRef Search ADS PubMed  45. Atucha E, Zalachoras I, van den Heuvel JK, van Weert LT, Melchers D, Mol IM, Belanoff JK, Houtman R, Hunt H, Roozendaal B, Meijer OC. A mixed glucocorticoid/mineralocorticoid selective modulator with dominant antagonism in the male rat brain. Endocrinology . 2015; 156( 11): 4105– 4114. Google Scholar CrossRef Search ADS PubMed  46. Zalachoras I, Houtman R, Atucha E, Devos R, Tijssen AM, Hu P, Lockey PM, Datson NA, Belanoff JK, Lucassen PJ, Joëls M, de Kloet ER, Roozendaal B, Hunt H, Meijer OC. Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator. Proc Natl Acad Sci USA . 2013; 110( 19): 7910– 7915. Google Scholar CrossRef Search ADS PubMed  47. Beck IM, Drebert ZJ, Hoya-Arias R, Bahar AA, Devos M, Clarisse D, Desmet S, Bougarne N, Ruttens B, Gossye V, Denecker G, Lievens S, Bracke M, Tavernier J, Declercq W, Gevaert K, Vanden Berghe W, Haegeman G, De Bosscher K. Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation. PLoS One . 2013; 8( 7): e69115. Google Scholar CrossRef Search ADS PubMed  48. van Lierop MJ, Alkema W, Laskewitz AJ, Dijkema R, van der Maaden HM, Smit MJ, Plate R, Conti PG, Jans CG, Timmers CM, van Boeckel CA, Lusher SJ, McGuire R, van Schaik RC, de Vlieg J, Smeets RL, Hofstra CL, Boots AM, van Duin M, Ingelse BA, Schoonen WG, Grefhorst A, van Dijk TH, Kuipers F, Dokter WH. Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index. PLoS One . 2012; 7( 11): e48385. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

Selective Glucocorticoid Receptor Antagonist CORT125281 Activates Brown Adipose Tissue and Alters Lipid Distribution in Male Mice

Loading next page...
 
/lp/ou_press/selective-glucocorticoid-receptor-antagonist-cort125281-activates-IUNOD00P5F
Publisher
Oxford University Press
Copyright
Copyright © 2018 Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
D.O.I.
10.1210/en.2017-00512
Publisher site
See Article on Publisher Site

Abstract

Abstract Glucocorticoids influence a wide range of metabolic processes in the human body, and excessive glucocorticoid exposure is known to contribute to the development of metabolic disease. We evaluated the utility of the novel glucocorticoid receptor (GR) antagonist CORT125281 for its potential to overcome adiposity, glucose intolerance, and dyslipidemia and compared this head-to-head with the classic GR antagonist RU486 (mifepristone). We show that, although RU486 displays cross-reactivity to the progesterone and androgen receptor, CORT125281 selectively inhibits GR transcriptional activity. In a mouse model for diet-induced obesity, rhythmicity of circulating corticosterone levels was disturbed. CORT125281 restored this disturbed rhythmicity, in contrast to RU486, which further inhibited endogenous corticosterone levels and suppressed adrenal weight. Both CORT125281 and RU486 reduced body weight gain and fat mass. In addition, CORT125281, but not RU486, lowered plasma levels of triglycerides, cholesterol, and free fatty acids and strongly stimulated triglyceride-derived fatty acid uptake by brown adipose tissue depots. In combination with reduced lipid content in brown adipocytes, this indicates that CORT125281 enhances metabolic activity of brown adipose tissue depots. CORT125281 was also found to increase liver lipid accumulation. Taken together, CORT125281 displayed a wide range of beneficial metabolic activities that are in part distinct from RU486, but clinical utility may be limited due to liver lipid accumulation. This warrants further evaluation of GR antagonists or selective modulators that are not accompanied by liver lipid accumulation while preserving their beneficial metabolic activities. Obesity and dyslipidemia constitute major problems in modern society (1, 2), and it is increasingly being recognized that glucocorticoid (GC) stress hormones contribute to such metabolic abnormalities (3). GCs are produced in the adrenal cortex and bind to the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR), thereby regulating a wide range of processes in the human body, including lipid and glucose mobilization and disposal. Circulating GC levels display a diurnal rhythmicity, and GCs are released in response to stress. Hypothalamic-pituitary-adrenal (HPA) axis activity regulates GC secretion by a cascade of hormonal processes, initiated by release of corticotropin-releasing hormone and vasopressin by the paraventricular nucleus of the hypothalamus, which results in secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary. ACTH subsequently stimulates GC production and secretion by the adrenals. HPA axis activity is controlled by GC-mediated negative feedback on multiple levels including the inhibition of ACTH release (4, 5). Hyperactivity of the HPA axis (e.g., in Cushing syndrome) causes a myriad of metabolic adverse effects, and GR antagonists were shown to be effective in counteracting this (6). Despite being extensively used in the clinic, GR antagonist RU486 (mifepristone) lacks receptor selectivity (7) and may in certain settings also exhibit partial agonist activity (8). Therefore, the use of a selective GR antagonist that lacks partial agonistic properties may be of value. Brown adipose tissue (BAT) is a relevant metabolic target tissue of GC that has been actively pursued to combat obesity and related disorders after its discovery in humans (9). BAT effectively combusts glucose and fatty acids into heat, contributing to energy expenditure (10). BAT is activated by cold via enhanced sympathetic outflow. The norepinephrine (NE) released from sympathetic nerve terminals binds to the β3-adrenergic receptor on brown adipocytes and strongly enhances activity and expression of uncoupling protein 1 (UCP-1), the main effector protein involved in thermogenesis (11). Therapeutic targeting of BAT (e.g., with a β3-adrenergic receptor agonist) may provide an effective strategy to improve metabolic health, as it alleviates dyslipidemia, lowers blood glucose, prevents weight gain, and protects from atherosclerosis development in mice (12, 13). Accumulating evidence indicates that chronic exposure to elevated endogenous GC (14, 15) or synthetic GR agonists (16) inhibits the activity of brown adipocytes and hampers the browning of white adipose tissue (WAT) (17), although acute effects may differ between mouse and man (18). Vice versa, the classic GR antagonist RU486 was shown to acutely stimulate BAT activity (8, 15, 19). In this study, we characterized novel GR antagonist CORT125281 (20) and evaluated its effects on energy metabolism and lipid distribution in male C57BL/6J mice fed a high-fat diet (HFD). CORT125281 effectively inhibited GR activity in several cell culture models, whereas MR, progesterone receptor (PR), and androgen receptor (AR) activity was unaffected. CORT125281 inhibited weight gain and lowered plasma lipids in a model for diet-induced obesity, accompanied by robust activation of BAT in comparison with RU486. CORT125281 adversely affected hepatic lipid metabolism in mice, warranting further search for selective GR modulators that efficiently antagonize GR in BAT without adversely affecting the liver. Materials and Methods Animals All animal studies reported here have been approved by the ethical committee of Leiden University Medical Center. Mice were housed in conventional cages with a 12:12-hour light-dark cycle with ad libitum access to food and water. Ten-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were fed a control chow diet vs chow diet supplemented with CORT125281 (60 mg/kg/d) for 3 weeks (n = 8 per group); received water containing 10% fructose in combination with an HFD (60% lard, Research Diets) supplemented with vehicle, CORT125281 (60 mg/kg/d), or RU486 (also known as mifepristone; 60 mg/kg/d) for 3 weeks (n = 8 per group); or were treated with different dosages of CORT125281 by diet supplementation (6, 20, or 60 mg/kg/d) for 3 weeks (n = 7 to 8 per group). Body weight, body composition, and indirect calorimetry measurements Body weight and composition (EchoMRI-100, Houston, TX) were determined throughout all experiments. Indirect calorimetry was performed in fully automatic metabolic cages (LabMaster System; TSE Systems, Bad Homburg, Germany) from day 1 until day 6. Oxygen consumption, carbohydrate production, and caloric intake were measured and used to calculate energy expenditure and fat and carbohydrate oxidation (21). Stress-free blood collection and corticosterone measurement Stress-free blood samples (i.e., drawn within 2 minutes before plasma corticosterone levels rise) were collected after 13 days in the morning (8:30, zeitgeber time 1.5) and evening (17:30, zeitgeber time 10.5), and corticosterone levels were determined using a 125I radioimmunoassay kit (ImmuChem; MP Biochemicals, Orangeburg, NY). Plasma lipid determination After 3 weeks of treatment, blood was collected from 4-hour fasted mice to determine plasma triglycerides (TGs), plasma total cholesterol (both with enzymatic kits from Roche Diagnostics, Mannheim, Germany), and plasma free fatty acids (FFAs) (NEFA C kit; Wako Diagnostics, Instruchemie, Delfzijl, The Netherlands). Intravenous glucose tolerance test After 2 weeks, a glucose tolerance test was performed. Mice were fasted for 6 hours, and at t = 0 minutes, blood was collected. After this, mice were intravenously injected with glucose (2 g/kg), and blood was collected at t = 5, 15, 30, 60, and 120 minutes. In all samples, plasma glucose was measured using an enzymatic kit (Instruchemie). TG clearance experiment At the end of the experiment, the clearance of TGs was determined. Glycerol tri[3H]oleate ([3H]TO)–labeled lipoprotein-like emulsion particles (1.0 mg TGs in 200 µL phosphate-buffered saline) were injected intravenously in the tail vein of the mice, and blood was collected at t = 2, 5, 10, and 15 minutes (22). Mice were euthanized by cervical dislocation directly after the last blood sample and perfused with ice-cold phosphate-buffered saline for 5 minutes, and organs were harvested, weighted, and divided in pieces for messenger RNA analysis, histology, or analysis of 3H activity. Histology Metabolic organs [i.e., interscapular BAT (iBAT), gonadal WAT, and liver] were fixated in 4% paraformaldehyde for 1 day and stored in 70% ethanol until further processing. Tissues were dehydrated and embedded in paraffin, and 5-µm sections were stained for hematoxylin-eosin and Oil Red O as previously described (23). Intracellular lipid droplet size and lipid content were quantified using Image J software (version 1.47). Cell culture HEK293T cells Human HEK293T cells were transfected using Fugene HD transfection reagent (Promega, Leiden, the Netherlands) with 25 ng TAT3-luciferase (TAT3-luc), 1 ng CAGGS-renilla, 100 ng pcDNA, and 10 ng human GR, MR, AR, or PR expression vector. Cells were pretreated with different concentrations of RU486 or CORT125281 for 1 hour before exposure to 50 nM cortisol (= hydrocorticosone) (for GR, 74-fold induction of GR signaling, data not shown), 10 nM cortisol (for MR, sixfold induction of MR signaling, data not shown), 10 nM progesterone (for PR, sixfold induction of PR signaling, data not shown), or 100 nM dihydrotestosterone (DHT) (for AR, fourfold induction of AR signaling, data not shown). After 24 hours, firefly and renilla luciferase signals were measured using a Dual Luciferase assay (Promega). Cell culture murine brown adipocytes Brown preadipocytes from murine BAT depots were isolated from 5-week-old male C57BL6/J mice. Cells were reversibly immortalized by using a lentiviral vector conferring doxycylin-controlled expression of simian virus large T antigen and expanded in maintenance medium (Dulbecco’s modified Eagle medium/F12 medium supplemented with heat-inactivated fetal bovine serum, 4.5 g/L glucose, penicillin/streptomycin, and 0.1 µg/mL doxycycline). Adipogenic differentiation was induced by culturing the cells for 13 to 15 days in differentiation medium (Dulbecco’s modified Eagle medium/F12 supplemented with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum, penicillin/streptomycin, 4 nM bovine insulin, 10 mM HEPES, 25 µg/mL ascorbate, and 1 µM rosiglitazone). During the last 2 days of differentiation and during the experiments, GC-free charcoal-stripped serum was used and the effects on GR transcriptional activity and BAT activity were examined in fully differentiated brown adipocytes. BAT activity was stimulated with 1 µM NE, and cells were simultaneously exposed to a combination of 10 to 1000 nM corticosterone, 10 to 1000 nM CORT125281, and/or 10 to 1000 nM RU486. After an incubation period of 8 hours, cells were lysed using TriPure (Roche, Mijdrecht, the Netherlands). RNA isolation, complementary DNA synthesis, and reverse transcription polymerase chain reaction analysis Total RNA was isolated using TriPure (Roche) according to the manufacturer’s protocol, and 500 to 1000 ng RNA was reverse-transcribed using M-MLV reverse-transcriptase (Promega). Reverse transcription polymerase chain reaction was performed on a CFX96 PCR machine using IQ SYBR-Green (Bio-Rad, Veenendaal, the Netherlands), and expression levels were normalized to housekeeping genes β2-microglobulin (B2M) or 36B4. Primer sequences are shown in Supplemental Table 1. Statistical analysis All data are presented as mean ± standard error of the mean. Statistical analyses were performed with GraphPad Prism 7 software (GraphPad Inc., La Jolla, CA), and for mixed-model analysis, IBM SPSS 23 software was used. Statistical differences were calculated with a one-way analysis of variance with the Tukey multiple-comparison test, with a two-way analysis of variance with the Tukey multiple-comparison test, with a linear mixed model with time as the covariate, or with an unpaired t test, as appropriate. P < 0.05 was considered significant for all analyses. IC50 values were calculated using GraphPad Prism 7 software, using a nonlinear fit model. Results CORT125281 selectively inhibits GR whereas RU486 exhibits cross-reactivity for PR and AR The effect of the novel GR antagonist CORT125281 on GR transcriptional activity was examined and compared with classic GR antagonist RU486. Human HEK293T cells were transfected with GR and a TAT3-luc reporter, and this revealed the expected inhibition of cortisol-induced GR activity by both GR antagonists (20), in which RU486 was significantly more potent than CORT125281 (IC50 of 43 nM and 427 nM, respectively, P < 0.0001) (Fig. 1A). To investigate receptor selectivity, HEK293T cells were transfected with MR, PR, or AR in combination with TAT3-luc and treated with their respective agonists around their estimated EC90 concentration (24). Neither CORT125281 nor RU486 affected cortisol-induced MR signaling (Fig. 1B). Although RU486 potently inhibited PR signaling (IC50: 0.6 nM) (Fig. 1C) and also displayed moderate inhibitory actions on AR signaling (IC50: 4.1 µM) (Fig. 1D), CORT125281 did not affect progesterone-induced PR signaling and DHT-induced AR signaling (Fig. 1C and 1D). Taken together, this supports the notion that CORT125281 is a selective GR antagonist, whereas RU486 exhibits cross-reactivity for PR and AR. Figure 1. View largeDownload slide The effect of GR antagonists on nuclear receptor signaling in vitro. HEK293T cells transfected with a TAT3-luc reporter were used to determine the antagonistic effects of CORT125281 and RU486 on (A) corticosterone-induced (10 nM) GR signaling, (B) corticosterone-induced (50 nM) MR signaling, (C) progesterone-induced (10 nM) PR signaling, and (D) DHT-induced (100 nM) AR signaling. Statistical significance was calculated using two-way analysis of variance with the Bonferroni multiple-comparisons test. **P < 0.01 vs CORT125281, ****P < 0.0001 vs CORT125281. Figure 1. View largeDownload slide The effect of GR antagonists on nuclear receptor signaling in vitro. HEK293T cells transfected with a TAT3-luc reporter were used to determine the antagonistic effects of CORT125281 and RU486 on (A) corticosterone-induced (10 nM) GR signaling, (B) corticosterone-induced (50 nM) MR signaling, (C) progesterone-induced (10 nM) PR signaling, and (D) DHT-induced (100 nM) AR signaling. Statistical significance was calculated using two-way analysis of variance with the Bonferroni multiple-comparisons test. **P < 0.01 vs CORT125281, ****P < 0.0001 vs CORT125281. CORT125281 reverses corticosterone-mediated GR activity in murine brown adipocytes in vitro To assess whether CORT125281 influences the activity of brown adipocytes, we used cell lines derived from murine BAT depots. Preadipocytes were differentiated into mature brown adipocytes and treated with corticosterone in combination with RU486 or CORT125281. To determine the effect of both compounds on GR transcriptional activity, we measured the expression of the well-known GR target genes Fkbp5 and Gilz (25, 26). Murine brown adipocytes were responsive to corticosterone, as treatment with 10 nM corticosterone significantly upregulated expression of Fkbp5 and Gilz (Fig. 2A and 2B). Treatment with either RU486 or CORT125281 effectively inhibited corticosterone-induced GR transcriptional activity (Fig. 2A and 2B). Also, for GR transcriptional activity induced by 1 µM corticosterone, both GR antagonists significantly inhibited GR target gene expression, although GR inhibition by 1 µM RU486 was stronger compared with 1 µM CORT125281, likely reflecting differences in binding affinity (Supplemental Fig. 1A and 1B) (20). As expected, activity of murine brown adipocytes was inhibited by corticosterone, that is, decreased NE-induced UCP-1 expression upon 10 nM (Fig. 2C) and 1 µM corticosterone exposure (Supplemental Fig. 1C). Both CORT125281 and RU486 were able to (partially) prevent corticosterone-induced inhibition of BAT activity, as coincubation with the GR antagonists results in enhanced UCP-1 expression (Fig. 2C, Supplemental Fig. 1C). Of note, RU486 did not dose-dependently reverse corticosterone-inhibited UCP-1 expression (Fig. 2C) and was not able, even at high doses, to fully prevent corticosterone-inhibited UCP-1 expression (Supplemental Fig. 1C). These findings may be explained by partial agonistic properties of RU486 on the GR. To test this, mature brown adipocytes were treated with different doses of RU486 or CORT125281, and this was compared with the agonistic effect of 10 nM corticosterone. Treatment with RU486 resulted in upregulation of the GR target gene Fkbp5 (P < 0.05) but not Gilz, whereas CORT125281 did not influence Fkbp5 or Gilz expression (Fig. 2D). RU486 treatment tended to reduce NE-induced Ucp1 expression (−40% vs vehicle), which is significant compared with 10 nM CORT125281 (P < 0.01, Fig. 2D). Taken together, these data suggest partial agonistic properties of RU486, which could limit BAT activating capacity by RU486, whereas CORT125281 showed only antagonistic properties on brown adipocytes. Figure 2. View largeDownload slide The effects of GR antagonists on murine BAT cells in vitro. RU486 and CORT125281 antagonistic properties on 10 nM corticosterone-regulated gene expression (A) Fkbp5 and (B) Gilz. **P < 0.01 vs NE, ***P < 0.001 vs NE, ****P < 0.0001 vs NE, $P < 0.05 vs CORT + NE, $$P < 0.01 vs CORT + NE, $$$$P < 0.0001 vs CORT + NE. (C) The effect of CORT125281 and RU486 on Ucp1 expression in murine brown adipocytes after 8 hours of exposure. ***P < 0.001, ****P < 0.0001, $$$P < 0.001 vs CORT, $$$$P < 0.0001 vs CORT, ^P < 0.05 vs CORT + NE, ^^P < 0.01 vs CORT + NE. (D) Assessment of partial agonism by RU486 and CORT125281 compared with 10 nM corticosterone on the expression of Fkbp5, Gilz, and UCP1 in murine brown adipocytes after 8 hours of exposure. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. CORT, corticosterone; Veh, vehicle. Figure 2. View largeDownload slide The effects of GR antagonists on murine BAT cells in vitro. RU486 and CORT125281 antagonistic properties on 10 nM corticosterone-regulated gene expression (A) Fkbp5 and (B) Gilz. **P < 0.01 vs NE, ***P < 0.001 vs NE, ****P < 0.0001 vs NE, $P < 0.05 vs CORT + NE, $$P < 0.01 vs CORT + NE, $$$$P < 0.0001 vs CORT + NE. (C) The effect of CORT125281 and RU486 on Ucp1 expression in murine brown adipocytes after 8 hours of exposure. ***P < 0.001, ****P < 0.0001, $$$P < 0.001 vs CORT, $$$$P < 0.0001 vs CORT, ^P < 0.05 vs CORT + NE, ^^P < 0.01 vs CORT + NE. (D) Assessment of partial agonism by RU486 and CORT125281 compared with 10 nM corticosterone on the expression of Fkbp5, Gilz, and UCP1 in murine brown adipocytes after 8 hours of exposure. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. CORT, corticosterone; Veh, vehicle. CORT125281 reduces body weight, fat mass, and plasma lipids in HFD-fed mice To evaluate the effects of CORT125281 on metabolism in a whole organism, a mouse model for diet-induced obesity was used. HFD-fed mice were treated with either CORT125281 (60 mg/kg/d) or the classic GR antagonist RU486 (60 mg/kg/d). For comparison, we also performed an experiment in which chow-fed mice were treated by diet supplementation with CORT125281 (60 mg/kg/d). At equal dosage, CORT125281 and RU486 similarly reduced HFD-induced body weight gain with ~10% (P < 0.001 for CORT125281 at day 21, Fig. 3A), whereas body weight was not altered in chow-fed mice (Supplemental Fig. 2A). Both GR antagonists significantly reduced fat mass but not lean mass in HFD-fed mice (−23% for RU486 and −32% for CORT125281 at day 21, P < 0.01 and P < 0.0001, respectively) (Fig. 3B and 3C), whereas CORT125281 did not affect fat mass or lean mass in chow-fed mice (Supplemental Fig. 2B and 2C). In the HFD condition, treatment with CORT125281 significantly lowered plasma TGs (−56%, P < 0.0001) (Fig. 3D) and cholesterol levels (−30%, P < 0.05) (Fig. 3E) compared with vehicle and RU486-treated mice, as well as significantly lowered plasma FFAs compared with RU486-treated mice (−23%, P < 0.05) (Fig. 3F). Similar plasma lipid-lowering activities of CORT125281 were found in chow-fed mice (i.e., significant reduction of plasma TGs and cholesterol and a near-significant reduction of FFAs) (Supplemental Fig. 2D–2F). In a subsequent experiment, different dosages of CORT125281 (6, 20, or 60 mg/kg/d) were evaluated in HFD-fed mice, which revealed that CORT125281 seemed to reduce body weight, fat mass, plasma TGs, cholesterol, and FFAs in a dose-dependent manner, with no effect on lean mass (Supplemental Fig. 3A–3F). In addition to lipid metabolism, we investigated the effects of CORT125281 on glucose metabolism. This revealed that CORT125281 did not affect basal glucose levels or intravenous glucose tolerance (Fig. 3G and 3H), whereas RU486 significantly improved glucose tolerance in HFD-fed mice, to a similar degree as previously described (27). Altogether, these data suggest that CORT125281 treatment reduces diet-induced weight gain and body fat mass and that CORT125281 effectively lowers plasma lipids. Figure 3. View largeDownload slide The effect of GR antagonists on body weight, body composition, and plasma lipids and glucose of HFD-fed C57BL/6J mice. The effect of the classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) body weight, (B) body fat mass, and (C) lean mass. The effect on (D) plasma TGs, (E) plasma total cholesterol (TC), and (F) plasma FFAs after 3 weeks of treatment. The effect on (G) basal glucose levels and (H) intravenous glucose tolerance (ivGTT) after 2 weeks of treatment. Statistical significance was calculated using (A–C) a mixed-model analysis, (D–G) a one-way analysis of variance with the Tukey multiple-comparisons test, or (H) a two-way analysis of variance with the Tukey multiple-comparisons test. ~P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 3. View largeDownload slide The effect of GR antagonists on body weight, body composition, and plasma lipids and glucose of HFD-fed C57BL/6J mice. The effect of the classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) body weight, (B) body fat mass, and (C) lean mass. The effect on (D) plasma TGs, (E) plasma total cholesterol (TC), and (F) plasma FFAs after 3 weeks of treatment. The effect on (G) basal glucose levels and (H) intravenous glucose tolerance (ivGTT) after 2 weeks of treatment. Statistical significance was calculated using (A–C) a mixed-model analysis, (D–G) a one-way analysis of variance with the Tukey multiple-comparisons test, or (H) a two-way analysis of variance with the Tukey multiple-comparisons test. ~P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CORT125281 restores HFD-disturbed HPA axis activity Under chow-fed conditions, mice display a circadian rhythm in circulating corticosterone with peak levels before the dark phase (i.e., start of the active period, data not shown). Three weeks of HFD resulted in dampening of the circadian corticosterone rhythm, as morning and evening circulating corticosterone levels were similar (Fig. 4A). RU486 treatment resulted in lower circulating corticosterone levels in the evening, suggesting an agonistic effect on the HPA axis resulting in enhanced negative feedback under this dose regimen (−80%, P < 0.01) (Fig. 4A). Strikingly, CORT125281 restored the HFD-disturbed circadian corticosterone rhythm (P < 0.0001, morning vs evening), and substantially higher circulating corticosterone levels in the evening were found compared with vehicle HFD-fed mice (P < 0.0001) (Fig. 4A). The lack of agonistic activity of CORT125281 is further supported by the observation that CORT125281 did not influence adrenal weight, whereas RU486 induced adrenal atrophy (−51% organ weight, P < 0.05) (Fig. 4B, Supplemental Fig. 4A), indicating continuous negative feedback on the HPA axis. At all evaluated dosages, CORT125281 did not influence the weight of the thymus and spleen (Fig. 4C and 4D, Supplemental Fig. 4B and 4C), organs that involute after chronic GC exposure, whereas RU486 was found to reduce thymus weight (−47%, P < 0.05) (Fig. 4C). Figure 4. View largeDownload slide The effect of GR antagonists on endogenous corticosterone and GC-sensitive organ weight in HFD-fed C57BL/6J mice. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) circulating corticosterone levels in the morning and evening and on (B) adrenal, (C) thymus, and (D) spleen weight. Statistical significance was calculated using a two-way analysis of variance with the (A) Tukey multiple-comparisons test or (B–D) a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, ****P < 0.0001, $$P < 0.01 vs vehicle in the evening, $$$$P < 0.0001 vs vehicle in the evening. AM, morning; NS, not significant; PM, evening; Veh, vehicle. Figure 4. View largeDownload slide The effect of GR antagonists on endogenous corticosterone and GC-sensitive organ weight in HFD-fed C57BL/6J mice. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) circulating corticosterone levels in the morning and evening and on (B) adrenal, (C) thymus, and (D) spleen weight. Statistical significance was calculated using a two-way analysis of variance with the (A) Tukey multiple-comparisons test or (B–D) a one-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, ****P < 0.0001, $$P < 0.01 vs vehicle in the evening, $$$$P < 0.0001 vs vehicle in the evening. AM, morning; NS, not significant; PM, evening; Veh, vehicle. CORT125281 stimulates fatty acid uptake and combustion by interscapular BAT To monitor energy expenditure, mice were housed in fully automated metabolic cages during the first week of treatment. RU486 significantly increased total energy expenditure, whereas CORT125281 did not (Fig. 5A). Treatment with RU486 and CORT125281 both resulted in increased fat oxidation (Fig. 5B) and decreased carbohydrate oxidation (Fig. 5C), as evident from a lowered respiratory exchange ratio (Fig. 5D). We next investigated the fate of intravenously injected lipoprotein-like particles labeled with [3H]TO lipids. In mice treated with CORT125281 but not RU486, plasma decay of [3H]TO was more rapid (P < 0.01, Fig. 5E), indicating enhanced TG uptake from plasma. Uptake of [3H]TO-derived activity by iBAT and dorso-cervical BAT was significantly increased in the CORT125281 group (+115%, P < 0.0001 and +61%, P < 0.05, respectively) (Fig. 5F), indicating enhanced metabolic activity of these BAT depots. This is further supported by a tendency toward reduced iBAT weight (Fig. 5G, Supplemental Fig. 5A), accompanied by reduced lipid content in CORT125281-treated mice compared with RU486-treated mice (−51%, P < 0.05) (Fig. 5H and 5I). In addition, CORT125281- and RU486-treated mice showed decreased gonadal WAT weight and smaller average cell size (Fig. 5J–5L, Supplemental Fig. 5B). Collectively, these data suggest that CORT125281 activated BAT to stimulate fatty acid uptake and combustion, whereas RU486 did not. Figure 5. View largeDownload slide The effect of GR antagonists on the activity of metabolic organs. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) energy expenditure (EE), (B) fat oxidation, (C) carbohydrate oxidation, and (D) respiratory exchange ratio. The effect on (E) plasma decay and (F) uptake of lipoprotein TG-derived FFA by metabolic tissues. The effect on (G) iBAT weight and (H) lipid content, as well as (I) representative images of hematoxylin and eosin–stained iBAT slices. The effect on (J) gWAT weight and (K) average cell size, as well as (L) representative images of hematoxylin and eosin–stained gWAT slices. Statistical significance was calculated using a (A–D, G, H, J, K) one-way analysis of variance with the Tukey multiple-comparisons test or (E, F) two-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$P < 0.01 vs RU486, $$$$P < 0.0001 vs RU486. dcBAT, dorsocervical BAT; gWAT, gonadal WAT; sWAT, subcutaneous white adipose tissue; Veh, vehicle; vWAT, visceral white adipose tissue. Figure 5. View largeDownload slide The effect of GR antagonists on the activity of metabolic organs. The effect of classic GR antagonist RU486 and the novel GR antagonist CORT125281 on (A) energy expenditure (EE), (B) fat oxidation, (C) carbohydrate oxidation, and (D) respiratory exchange ratio. The effect on (E) plasma decay and (F) uptake of lipoprotein TG-derived FFA by metabolic tissues. The effect on (G) iBAT weight and (H) lipid content, as well as (I) representative images of hematoxylin and eosin–stained iBAT slices. The effect on (J) gWAT weight and (K) average cell size, as well as (L) representative images of hematoxylin and eosin–stained gWAT slices. Statistical significance was calculated using a (A–D, G, H, J, K) one-way analysis of variance with the Tukey multiple-comparisons test or (E, F) two-way analysis of variance with the Tukey multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$P < 0.01 vs RU486, $$$$P < 0.0001 vs RU486. dcBAT, dorsocervical BAT; gWAT, gonadal WAT; sWAT, subcutaneous white adipose tissue; Veh, vehicle; vWAT, visceral white adipose tissue. CORT125281 increases liver lipid content As GR activity is known to influence hepatic function (e.g., lipid uptake, very-low-density lipoprotein production, de novo lipogenesis) (14), we next analyzed the livers of mice treated with RU486 and CORT125281. In HFD-fed mice, CORT125281 significantly increased liver weight (+51%, P < 0.01) (Fig. 6A, Supplemental Fig. 5C) and treatment with CORT125281 was accompanied by increased liver TGs (+88%, P < 0.05) (Supplemental Fig. 5D) and total liver lipids (visualized by an Oil Red O staining, Fig. 6B and 6C). Also, in chow-fed mice, CORT125281 seemed to increase liver weight and significantly increased liver TGs (Supplemental Fig. 2G and 2H). Of note, both RU486 and CORT125281 strongly upregulated expression of Cyp3a11, suggestive of induction of the pregnane X receptor (PXR) (Fig. 6D). PXR agonism is known to cause hepatic lipid accumulation, at least partly via increased hepatic expression of fatty acid transporter Cd36 (28), which we also observed for both CORT125281 and RU486 (Fig. 6E). Although RU486 induced Cyp3a11 expression most strongly, this was not accompanied by the highest hepatic lipid content (Fig. 6B–6E), suggesting additional, differential effects of RU486 and CORT125281 on liver lipid metabolism. Figure 6. View largeDownload slide The effect of GR antagonists on the liver. The effect of the novel GR antagonist CORT125281 and the classic GR antagonist RU486 on (A) liver weight and (B) liver Oil Red O. (C) Representative images of Oil Red O–stained liver slices. Hepatic expression of (D) Cyp3a11 and (E) Cd36. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. **P < 0.01 vs Veh, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$$$P < 0.0001 vs RU486. Veh, vehicle. Figure 6. View largeDownload slide The effect of GR antagonists on the liver. The effect of the novel GR antagonist CORT125281 and the classic GR antagonist RU486 on (A) liver weight and (B) liver Oil Red O. (C) Representative images of Oil Red O–stained liver slices. Hepatic expression of (D) Cyp3a11 and (E) Cd36. Statistical significance was calculated using a one-way analysis of variance with the Tukey multiple-comparisons test. **P < 0.01 vs Veh, ****P < 0.0001 vs Veh, $P < 0.05 vs RU486, $$$$P < 0.0001 vs RU486. Veh, vehicle. Discussion In this study, we describe the effects of a novel GR antagonist CORT125281 on metabolism and HPA axis activity in a model for diet-induced obesity in male mice. In our studies, we compared CORT125281 head-to-head with the classic GR antagonist RU486. First, we characterized the GR specificity of CORT125281, and luciferase reporter experiments clearly show that CORT125281 selectively inhibits the GR, whereas the classic GR antagonist RU486 also inhibits PR and AR transcriptional activity, as expected (7, 29). In a model for diet-induced obesity, we have shown that CORT125281 and RU486 equally reduce body weight gain in HFD-fed mice, similar to previous observations for RU486 (30). In addition, both GR antagonists reduce total fat mass without adversely affecting the lean mass. CORT125281, but not RU486, significantly lowered plasma lipids, restored circadian corticosterone rhythmicity, and induced fatty acid uptake and combustion by iBAT. Both GR antagonists effectively reversed corticosterone-suppressed UCP-1 expression in brown adipocytes in vitro, and this reversal of corticosterone-inhibitory actions on BAT could partially underlie the CORT125281-induced BAT activity observed in vivo. Although RU486 may activate BAT in specific contexts (8), in the present setting, RU486 did not seem to activate BAT in vivo, and this discrepancy between RU486 and CORT125281 may be explained by differential effects on PR or AR activity. Alternatively, because ACTH was shown to stimulate BAT activity (15), the restored HPA axis activity in CORT125281-treated mice may augment circulating ACTH levels and thereby enhance ACTH-induced BAT activation. The lack of partial agonism on the HPA axis by CORT125281 may thus underlie the differential effects of RU486 and CORT125281 on BAT activity in vivo. Additional partial agonistic activities of RU486 (as evident from reduced NE-induced UCP-1 expression in RU486-treated brown adipocytes in vitro, Fig. 2D) may also explain the lower BAT-activating capacity of RU486. In our study, we observed disturbed corticosterone rhythmicity upon HFD, which is in line with a previous study (31). Nutrient sensors influence the peripheral clock (32), and HFD was shown to alter diurnal patterns of leptin and insulin, as well as to reduce circadian patterns of clock genes in metabolic tissues (33). Thus, the flattened corticosterone rhythm observed in our study could be a consequence of HFD-disturbed circadian rhythm, which is supported by the observation that the HFD-fed mice in our study eat throughout the whole day rather than mainly in the dark period (data not shown). Alternatively, HFD could influence the HPA axis and its hormones directly, as decreased 11β-HSD1 expression (which converts inactive into active GC) and altered corticotropin-releasing hormone and GR expression in the paraventricular nucleus were observed upon HFD feeding (31). Fatty acids were also shown to regulate circulating corticosterone levels, and fatty acid sensors are known to be present in the hypothalamus (34). FFA-lowering strategies (e.g., insulin administration) were shown to increase plasma ACTH and corticosterone levels (35), and based on this, the decreased plasma FFA levels upon CORT125281 treatment could contribute to the restored corticosterone rhythmicity observed in our study. In addition, the lack of peripheral negative GC feedback on the HPA axis, due to the continuous presence of CORT125281, could contribute to the restored corticosterone rhythmicity. Although acute RU486 treatment can interfere with GR-mediated negative feedback and disinhibit the HPA axis (36), in the present setting (continuous administration of high dose via the food), both corticosterone levels and adrenal weights were strongly reduced, suggesting suppression of ACTH release rather than classic disinhibition. Although the reduced plasma lipids upon CORT125281 treatment can partially be attributed to enhanced BAT activity, it seems likely that enhanced lipid uptake by the liver is also involved. HFD induces hepatic expression of the cellular fatty acid transporter CD36 (37). CD36 mediates hepatic lipid uptake and is critically involved in the pathogenesis of liver steatosis as its upregulation induces lipid accumulation in the liver (38), and hepatic deletion of CD36 prevents this (39). Both endogenous GC (14, 40) and synthetic GC agonists (41, 42) have been shown to increase hepatic CD36 expression, thereby aggravating liver steatosis (14). Vice versa, GR knockout decreases hepatic CD36 expression and subsequently lowers liver lipids (43). Surprisingly, treatment with CORT125281 and RU486 also increased hepatic Cd36 expression, which is likely attributed to activation of the xenobiotic sensor PXR (44). This subsequently enhances fatty acid uptake, resulting in lipid accumulation and enhanced liver weight in mice. Currently, it is unknown if CORT125281 induces similar hepatic lipid accumulation in humans. Remarkably, in our studies, RU486 treatment is not associated with lipid accumulation, whereas RU486 is a known PXR ligand (28) and enhanced hepatic Cd36 and Cyp3a11 expression. This suggests additional lipid-lowering activities of RU486 (e.g., very-low-density lipoprotein production, β-oxidation) that prevent hepatic lipid uptake and accumulation and the development of steatosis. The differential effects of RU486 and CORT125281 may therefore be a consequence of the partial agonistic features of RU486 that are lacking in CORT125281. To date, the utility of GR antagonists could be further improved for the treatment of metabolic disease. Based on our current study, GR antagonism with RU486 affects only body weight and fat mass but does not display additional beneficial metabolic activities, whereas the potential of CORT125281 may be limited due to adverse liver steatosis–inducing effects that we observe in mice. This may call for GR ligands that selectively act on BAT or that exhibit mixed agonistic and antagonistic features (45–48), to exploit the beneficial metabolic effects of both GR agonism and GR antagonism. Abbreviations: ACTH adrenocorticotropic hormone AR androgen receptor BAT brown adipose tissue DHT dihydrotestosterone FFA free fatty acid GC glucocorticoid GR glucocorticoid receptor HFD high-fat diet HPA hypothalamic-pituitary-adrenal iBAT interscapular brown adipose tissue MR mineralocorticoid receptor NE norepinephrine PR progesterone receptor PXR pregnane X receptor TAT3-luc TAT3-luciferase TG triglyceride UCP-1 uncoupling protein 1 WAT white adipose tissue. Acknowledgments We thank Trea Streefland for valuable technical assistance; Jia Liu and Antoine de Vries from Leiden University Medical Center’s Laboratory of Experimental Cardiology, as well as Sander Kooijman from the Department of Medicine, for generating lines of reversibly immortalized murine brown preadipocytes; and Dr. A. O. Brinkmann for providing the human PR and AR constructs. Financial Support: This study was partially funded by Corcept Therapeutics. L.L.K. was funded with a grant by the Board of Directors of Leiden University Medical Center. Disclosure Summary: Hazel Hunt is an employee of Corcept Therapeutics, a company that develops selective receptor modulators, including CORT125281. The remaining authors have nothing to disclose. References 1. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. JAMA . 2007; 298( 3): 299– 308. Google Scholar CrossRef Search ADS PubMed  2. Phillips CM, Tierney AC, Perez-Martinez P, Defoort C, Blaak EE, Gjelstad IM, Lopez-Miranda J, Kiec-Klimczak M, Malczewska-Malec M, Drevon CA, Hall W, Lovegrove JA, Karlstrom B, Risérus U, Roche HM. Obesity and body fat classification in the metabolic syndrome: impact on cardiometabolic risk metabotype. Obesity (Silver Spring) . 2013; 21( 1): E154– E161. Google Scholar CrossRef Search ADS PubMed  3. Walker BR. Cortisol—cause and cure for metabolic syndrome? Diabet Med . 2006; 23( 12): 1281– 1288. Google Scholar CrossRef Search ADS PubMed  4. Shipston MJ. Mechanism(s) of early glucocorticoid inhibition of adrenocorticotropin secretion from anterior pituitary corticotropes. Trends Endocrinol Metab . 1995; 6( 8): 261– 266. Google Scholar CrossRef Search ADS PubMed  5. Herman JP, McKlveen JM, Solomon MB, Carvalho-Netto E, Myers B. Neural regulation of the stress response: glucocorticoid feedback mechanisms. Braz J Med Biol Res . 2012; 45( 4): 292– 298. Google Scholar CrossRef Search ADS PubMed  6. Castinetti F, Brue T, Conte-Devolx B. The use of the glucocorticoid receptor antagonist mifepristone in Cushing’s syndrome. Curr Opin Endocrinol Diabetes Obes . 2012; 19( 4): 295– 299. Google Scholar CrossRef Search ADS PubMed  7. Gaillard RC, Riondel A, Muller AF, Herrmann W, Baulieu EE. RU 486: a steroid with antiglucocorticosteroid activity that only disinhibits the human pituitary-adrenal system at a specific time of day. Proc Natl Acad Sci USA . 1984; 81( 12): 3879– 3882. Google Scholar CrossRef Search ADS PubMed  8. van den Heuvel JK, Boon MR, van Hengel I, Peschier-van der Put E, van Beek L, van Harmelen V, van Dijk KW, Pereira AM, Hunt H, Belanoff JK, Rensen PC, Meijer OC. Identification of a selective glucocorticoid receptor modulator that prevents both diet-induced obesity and inflammation. Br J Pharmacol . 2016; 173( 11): 1793– 1804. Google Scholar CrossRef Search ADS PubMed  9. Poekes L, Lanthier N, Leclercq IA. Brown adipose tissue: a potential target in the fight against obesity and the metabolic syndrome. Clin Sci (Lond) . 2015; 129( 11): 933– 949. Google Scholar CrossRef Search ADS PubMed  10. Kooijman S, van den Heuvel JK, Rensen PC. Neuronal control of brown fat activity. Trends Endocrinol Metab . 2015; 26( 11): 657– 668. Google Scholar CrossRef Search ADS PubMed  11. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev . 2004; 84( 1): 277– 359. Google Scholar CrossRef Search ADS PubMed  12. Warner A, Kjellstedt A, Carreras A, Böttcher G, Peng XR, Seale P, Oakes N, Lindén D. Activation of β3-adrenoceptors increases in vivo free fatty acid uptake and utilization in brown but not white fat depots in high-fat-fed rats. Am J Physiol Endocrinol Metab . 2016; 311( 6): E901– E910. Google Scholar CrossRef Search ADS PubMed  13. Berbée JF, Boon MR, Khedoe PP, Bartelt A, Schlein C, Worthmann A, Kooijman S, Hoeke G, Mol IM, John C, Jung C, Vazirpanah N, Brouwers LP, Gordts PL, Esko JD, Hiemstra PS, Havekes LM, Scheja L, Heeren J, Rensen PC. Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development. Nat Commun . 2015; 6: 6356. Google Scholar CrossRef Search ADS PubMed  14. van den Beukel JC, Boon MR, Steenbergen J, Rensen PC, Meijer OC, Themmen AP, Grefhorst A. Cold exposure partially corrects disturbances in lipid metabolism in a male mouse model of glucocorticoid excess. Endocrinology . 2015; 156( 11): 4115– 4128. Google Scholar CrossRef Search ADS PubMed  15. van den Beukel JC, Grefhorst A, Quarta C, Steenbergen J, Mastroberardino PG, Lombès M, Delhanty PJ, Mazza R, Pagotto U, van der Lely AJ, Themmen AP. Direct activating effects of adrenocorticotropic hormone (ACTH) on brown adipose tissue are attenuated by corticosterone. FASEB J . 2014; 28( 11): 4857– 4867. Google Scholar CrossRef Search ADS PubMed  16. Soumano K, Desbiens S, Rabelo R, Bakopanos E, Camirand A, Silva JE. Glucocorticoids inhibit the transcriptional response of the uncoupling protein-1 gene to adrenergic stimulation in a brown adipose cell line. Mol Cell Endocrinol . 2000; 165( 1–2): 7– 15. Google Scholar CrossRef Search ADS PubMed  17. Kong X, Yu J, Bi J, Qi H, Di W, Wu L, Wang L, Zha J, Lv S, Zhang F, Li Y, Hu F, Liu F, Zhou H, Liu J, Ding G. Glucocorticoids transcriptionally regulate miR-27b expression promoting body fat accumulation via suppressing the browning of white adipose tissue. Diabetes . 2014; 64( 2): 393– 404. Google Scholar CrossRef Search ADS PubMed  18. Ramage LE, Akyol M, Fletcher AM, Forsythe J, Nixon M, Carter RN, van Beek EJ, Morton NM, Walker BR, Stimson RH. Glucocorticoids acutely increase brown adipose tissue activity in humans, revealing species-specific differences in UCP-1 regulation. Cell Metab . 2016; 24( 1): 130– 141. Google Scholar CrossRef Search ADS PubMed  19. Rodríguez AM, Palou A. The steroid RU486 induces UCP1 expression in brown adipocytes. Pflugers Arch . 2004; 449( 2): 170– 174. Google Scholar CrossRef Search ADS PubMed  20. Hunt HJ, Belanoff JK, Walters I, Gourdet B, Thomas J, Barton N, Unitt J, Phillips T, Swift D, Eaton E. Identification of the clinical candidate (R)-(1-(4-Fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone (CORT125134): a selective glucocorticoid receptor (GR) antagonist. J Med Chem . 2017; 60( 8): 3405– 3421. Google Scholar CrossRef Search ADS PubMed  21. Van Klinken JB, van den Berg SA, Havekes LM, Willems Van Dijk K. Estimation of activity related energy expenditure and resting metabolic rate in freely moving mice from indirect calorimetry data. PLoS One . 2012; 7( 5): e36162. Google Scholar CrossRef Search ADS PubMed  22. Rensen PC, Herijgers N, Netscher MH, Meskers SC, van Eck M, van Berkel TJ. Particle size determines the specificity of apolipoprotein E–containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo. J Lipid Res . 1997; 38( 6): 1070– 1084. Google Scholar PubMed  23. Kooijman S, Boon MR, Parlevliet ET, Geerling JJ, van de Pol V, Romijn JA, Havekes LM, Meurs I, Rensen PC. Inhibition of the central melanocortin system decreases brown adipose tissue activity. J Lipid Res . 2014; 55( 10): 2022– 2032. Google Scholar CrossRef Search ADS PubMed  24. Kizu R, Otsuki N, Kishida Y, Toriba A, Mizokami A, Burnstein KL, Klinge CM, Hayakawai K. A new luciferase reporter gene assay for the detection of androgenic and antiandrogenic effects based on a human prostate specific antigen promoter and PC3/AR human prostate cancer cells. Anal Sci . 2004; 20( 1): 55– 59. Google Scholar CrossRef Search ADS PubMed  25. Zalachoras I, Verhoeve SL, Toonen LJ, van Weert LT, van Vlodrop AM, Mol IM, Meelis W, de Kloet ER, Meijer OC. Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression. Mol Psychiatry . 2016; 21( 12): 1733– 1739. Google Scholar CrossRef Search ADS PubMed  26. Drebert Z, Bracke M, Beck IM. Glucocorticoids and the non-steroidal selective glucocorticoid receptor modulator, compound A, differentially affect colon cancer–derived myofibroblasts. J Steroid Biochem Mol Biol . 2015; 149: 92– 105. Google Scholar CrossRef Search ADS PubMed  27. Mammi C, Marzolla V, Armani A, Feraco A, Antelmi A, Maslak E, Chlopicki S, Cinti F, Hunt H, Fabbri A, Caprio M. A novel combined glucocorticoid-mineralocorticoid receptor selective modulator markedly prevents weight gain and fat mass expansion in mice fed a high-fat diet. Int J Obes . 2016; 40( 6): 964– 972. Google Scholar CrossRef Search ADS   28. Zhou C, King N, Chen KY, Breslow JL. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice. J Lipid Res . 2009; 50( 10): 2004– 2013. Google Scholar CrossRef Search ADS PubMed  29. Schreiber JR, Hsueh AJ, Baulieu EE. Binding of the anti-progestin RU-486 to rat ovary steroid receptors. Contraception . 1983; 28( 1): 77– 85. Google Scholar CrossRef Search ADS PubMed  30. Asagami T, Belanoff JK, Azuma J, Blasey CM, Clark RD, Tsao PS. Selective glucocorticoid receptor (GR-II) antagonist reduces body weight gain in mice. J Nutr Metab . 2011; 2011: 235389. Google Scholar CrossRef Search ADS PubMed  31. Auvinen HE, Romijn JA, Biermasz NR, Pijl H, Havekes LM, Smit JW, Rensen PC, Pereira AM. The effects of high fat diet on the basal activity of the hypothalamus-pituitary-adrenal axis in mice. J Endocrinol . 2012; 214( 2): 191– 197. Google Scholar CrossRef Search ADS PubMed  32. Oosterman JE, Kalsbeek A, la Fleur SE, Belsham DD. Impact of nutrients on circadian rhythmicity. Am J Physiol Regul Integr Comp Physiol . 2014; 308( 5): R337– R350. Google Scholar CrossRef Search ADS PubMed  33. Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y, Turek FW, Bass J. High-fat diet disrupts behavioral and molecular circadian rhythms in mice. Cell Metab . 2007; 6( 5): 414– 421. Google Scholar CrossRef Search ADS PubMed  34. Lam TK, Schwartz GJ, Rossetti L. Hypothalamic sensing of fatty acids. Nat Neurosci . 2005; 8( 5): 579– 584. Google Scholar CrossRef Search ADS PubMed  35. Oh YT, Oh KS, Kang I, Youn JH. A fall in plasma free fatty acid (FFA) level activates the hypothalamic-pituitary-adrenal axis independent of plasma glucose: evidence for brain sensing of circulating FFA. Endocrinology . 2012; 153( 8): 3587– 3592. Google Scholar CrossRef Search ADS PubMed  36. Ratka A, Sutanto W, Bloemers M, de Kloet ER. On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation. Neuroendocrinology . 1989; 50( 2): 117– 123. Google Scholar CrossRef Search ADS PubMed  37. Sheedfar F, Sung MM, Aparicio-Vergara M, Kloosterhuis NJ, Miquilena-Colina ME, Vargas-Castrillón J, Febbraio M, Jacobs RL, de Bruin A, Vinciguerra M, García-Monzón C, Hofker MH, Dyck JR, Koonen DP. Increased hepatic CD36 expression with age is associated with enhanced susceptibility to nonalcoholic fatty liver disease. Aging (Albany NY) . 2014; 6( 4): 281– 295. Google Scholar CrossRef Search ADS PubMed  38. Yao L, Wang C, Zhang X, Peng L, Liu W, Zhang X, Liu Y, He J, Jiang C, Ai D, Zhu Y. Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice. Hepatology . 2016; 64( 1): 92– 105. Google Scholar CrossRef Search ADS PubMed  39. Wilson CG, Tran JL, Erion DM, Vera NB, Febbraio M, Weiss EJ. Hepatocyte-specific disruption of CD36 attenuates fatty liver and improves insulin sensitivity in HFD-fed mice. Endocrinology . 2016; 157( 2): 570– 585. Google Scholar CrossRef Search ADS PubMed  40. Bowles NP, Karatsoreos IN, Li X, Vemuri VK, Wood JA, Li Z, Tamashiro KL, Schwartz GJ, Makriyannis AM, Kunos G, Hillard CJ, McEwen BS, Hill MN. A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome. Proc Natl Acad Sci USA . 2014; 112( 1): 285– 290. Google Scholar CrossRef Search ADS PubMed  41. Poggioli R, Ueta CB, Drigo RA, Castillo M, Fonseca TL, Bianco AC. Dexamethasone reduces energy expenditure and increases susceptibility to diet-induced obesity in mice. Obesity (Silver Spring) . 2013; 21( 9): E415– E420. Google Scholar PubMed  42. Feng B, He Q, Xu H. FOXO1-dependent up-regulation of MAP kinase phosphatase 3 (MKP-3) mediates glucocorticoid-induced hepatic lipid accumulation in mice. Mol Cell Endocrinol . 2014; 393( 1–2): 46– 55. Google Scholar CrossRef Search ADS PubMed  43. Lemke U, Krones-Herzig A, Berriel Diaz M, Narvekar P, Ziegler A, Vegiopoulos A, Cato AC, Bohl S, Klingmüller U, Screaton RA, Müller-Decker K, Kersten S, Herzig S. The glucocorticoid receptor controls hepatic dyslipidemia through Hes1. Cell Metab . 2008; 8( 3): 212– 223. Google Scholar CrossRef Search ADS PubMed  44. Bitter A, Rümmele P, Klein K, Kandel BA, Rieger JK, Nüssler AK, Zanger UM, Trauner M, Schwab M, Burk O. Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Arch Toxicol . 2014; 89( 11): 2089– 2103. Google Scholar CrossRef Search ADS PubMed  45. Atucha E, Zalachoras I, van den Heuvel JK, van Weert LT, Melchers D, Mol IM, Belanoff JK, Houtman R, Hunt H, Roozendaal B, Meijer OC. A mixed glucocorticoid/mineralocorticoid selective modulator with dominant antagonism in the male rat brain. Endocrinology . 2015; 156( 11): 4105– 4114. Google Scholar CrossRef Search ADS PubMed  46. Zalachoras I, Houtman R, Atucha E, Devos R, Tijssen AM, Hu P, Lockey PM, Datson NA, Belanoff JK, Lucassen PJ, Joëls M, de Kloet ER, Roozendaal B, Hunt H, Meijer OC. Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator. Proc Natl Acad Sci USA . 2013; 110( 19): 7910– 7915. Google Scholar CrossRef Search ADS PubMed  47. Beck IM, Drebert ZJ, Hoya-Arias R, Bahar AA, Devos M, Clarisse D, Desmet S, Bougarne N, Ruttens B, Gossye V, Denecker G, Lievens S, Bracke M, Tavernier J, Declercq W, Gevaert K, Vanden Berghe W, Haegeman G, De Bosscher K. Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation. PLoS One . 2013; 8( 7): e69115. Google Scholar CrossRef Search ADS PubMed  48. van Lierop MJ, Alkema W, Laskewitz AJ, Dijkema R, van der Maaden HM, Smit MJ, Plate R, Conti PG, Jans CG, Timmers CM, van Boeckel CA, Lusher SJ, McGuire R, van Schaik RC, de Vlieg J, Smeets RL, Hofstra CL, Boots AM, van Duin M, Ingelse BA, Schoonen WG, Grefhorst A, van Dijk TH, Kuipers F, Dokter WH. Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index. PLoS One . 2012; 7( 11): e48385. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society

Journal

EndocrinologyOxford University Press

Published: Jan 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off