The selective glucocorticoid receptor antagonist CORT125281 has tissue-specific activity

Abstract

Glucocorticoids mediate numerous essential processes in the human body via binding to the glucocorticoid receptor (GR). Excessive GR signaling can cause disease, and GR antagonists can be used to treat many symptoms of glucocorticoid-induced pathology. The purpose of this study was to characterize the tissue-specific properties of the selective GR antagonist CORT125281. We evaluated the antagonistic effects of CORT125281 upon acute and subchronic corticosterone exposure in mice. In the acute corticosterone setting, hypothalamus-pituitary-adrenal-axis activity was investigated by measurement of basal- and stress-induced corticosterone levels, adrenocorticotropic hormone levels and pituitary proopiomelanocortin expression. GR signaling was evaluated by RT-PCR analysis of GR-responsive transcripts in liver, muscle, brown adipose tissue (BAT), white adipose tissue (WAT) and hippocampus. Pretreatment with a high dose of CORT125281 antagonized GR activity in a tissue-dependent manner. We observed complete inhibition of GR-induced target gene expression in the liver, partial blockade in muscle and BAT and no antagonism in WAT and hippocampus. Tissue distribution only partially explained the lack of effective antagonism. CORT125281 treatment did not disinhibit the hypothalamus-pituitary-adrenal neuroendocrine axis. In the subchronic corticosterone setting, CORT125281 partially prevented corticosterone-induced hyperinsulinemia, but not hyperlipidemia and immune suppression. In conclusion, CORT125281 antagonizes GR transcriptional activity in a tissue-dependent manner and improves corticosterone-induced hyperinsulinemia. Tailored dosing of CORT125281 may allow tissue-specific inhibition of GR transcriptional activity.

Keywords: GR antagonist; HPA-axis; RU486; coregulators; corticosterone; glucocorticoid receptor; mifepristone; tissue-specificity.

Figure 1

CORT125281 exhibits tissue-specific GR antagonism. Mice received vehicle or 60 mg/kg/day CORT125281 for 6 days and received a solvent or corticosterone injection 1 h prior to killing. (A) In the liver, CORT125281 fully blocked the corticosterone-induced upregulation of general GR target genes Fkbp5, Gilz, Sgk1 and Mt2a and liver-specific GR target genes G6pc and Pepck. CORT125281 inhibited a subset of genes in (B) quadriceps muscle and (C) brown adipose tissue (BAT) and did not show efficacy in (D) white adipose tissue (WAT) and (E) hippocampus. Values are means ± s.e.m. of n = 7 mice per group. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

CORT125281 inhibits GR transcriptional activity in a dose- and tissue-dependent manner. Mice received vehicle or 6–20 mg/kg/day CORT125281 for 6 days and received a solvent or corticosterone injection 1 h prior to killing. CORT125281 partially prevented the corticosterone-induced upregulation of (A, B, C and D) Fkbp5, Gilz, Sgk1 and Mt2a in the liver. CORT125281 did not antagonize GR transcriptional activity in (E, F, G and H) brown adipose tissue (BAT) and (I, J, K and L) white adipose tissue (WAT). Values are means ± s.e.m. of n = 7–8 mice per group. Statistical significance was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, ***P < 0.001.

Figure 3

CORT125281 does not antagonize Gilz and Fkbp5 expression in the brain during excess corticosterone exposure. Mice received vehicle or 20 mg/kg/day CORT125281 for 6 days and received a vehicle or corticosterone injection 1 h prior to killing. (A, B and C) Corticosterone increased expression levels of Gilz (red) mRNA in CA1 and CA2 regions of the hippocampus and of Fkbp5 hnRNA (white) in the CA2, which was not prevented by CORT125281. (D, E and F) In the paraventricular nucleus of the hypothalamus (PVN), corticosterone did not affect Gilz (red) expression, but tended to induce Fkbp5 (white) hnRNA expression, which was not prevented by CORT125281. Values are means ± s.e.m. of n = 7–8 mice per group. Statistical significance was calculated using linear mixed models. *P < 0.05. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0486.

Figure 4

CORT125281 does not inhibit corticosterone-induced Nr3c1 and Nr3c2 expression in the brain. Mice received vehicle or 20 mg/kg/day CORT125281 for 6 days and received a vehicle or corticosterone injection 1 h prior to killing. (A, B and C) Neither corticosterone nor CORT125281 affected Nr3c1 (Gr, yellow) mRNA levels in the hippocampus, but CORT125281 upregulated Nr3c2 (Mr, magenta) mRNA levels in all regions of the hippocampus. (J, K and L) Neither corticosterone nor CORT125281 affected Nr3c1 (yellow) or Nr3c2 (magenta) expression in the paraventricular nucleus of the hypothalamus (PVN). Values are means ± s.e.m. of n = 7–8 mice per group. Statistical significance was calculated using linear mixed models. *P < 0.05. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0486.

Figure 5

CORT125281 does not influence basal- or stress-induced HPA-axis activity. Mice received vehicle or 6–20 mg/kg/day CORT125281 for 6 days and received a solvent or corticosterone injection 1 h prior to killing. (A) CORT125281 did not alter basal ACTH levels at 18:00 h. Statistical significance was calculated with a one-way ANOVA with Tukey’s multiple comparisons test. (B and C) CORT125281 did not affect Pomc expression (red) in the anterior pituitary (white arrows). (D) CORT125281 did not influence basal corticosterone levels at the trough (08:00 h) and peak (18:00 h). Statistical significance was calculated using linear mixed models. (E) CORT125281 did not alter novelty stress-induced corticosterone levels at day 2. (F) Weight of GC-responsive adrenals was unaltered upon CORT125281 treatment. Statistical significance was calculated with a two-way ANOVA with Tukey’s multiple comparisons test. (G and H) In mice treated with vehicle or 60 mg/kg/day CORT125281, CORT125281 did not alter basal corticosterone levels at the trough (08:00 h) and peak (18:00 h). Statistical significance was calculated with a two-way ANOVA. (H, I and J) While adrenal weights were unaltered by CORT125281 treatment, peak corticosterone levels after a novelty stressor and the area under the curve (AUC) tended to be reduced. Statistical significance was calculated with an independent sample t-test. Values are means ± s.e.m. of n = 6–8 per group. *** P < 0.001. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0486.

Figure 6

CORT125281 tissue-distribution or coregulator recruitment does not fully explain tissue-specific GR antagonism. Mice received 60 mg/kg/day CORT125281 for 6 days and plasma, liver, brown adipose tissue (BAT), gonadal white adipose tissue (gWAT) and the neocortex of the brain were collected to determine CORT125281 concentrations via mass spectrometry. Concentrations are expressed as (A) absolute tissue levels and as (B) tissue to plasma ratio. Values are means ± s.e.m. of n = 7 mice per group. (C) To investigate which coregulators were recruited by CORT125281, the ligand-induced interactions with coregulator motifs were identified with a MARcoNI assay (microarray assay for real-time coregulator-nuclear receptor interaction) and quantified as modulation index (MI, log fold change relative to vehicle). The heatmap shows hierarchically clustered modulation indexes for the applied ligands dexamethasone (top row), cortisol (second row), mifepristone (third row) and CORT125281 (bottom row). Stars indicate significance relative to vehicle. Some clusters are shown in more detail; (D) Cluster which includes corepressors NCOR1 and -2. (E) Motifs exclusively bound by the dexamethasone-GR complex. (F) Motifs for which interactions are induced by both dexamethasone and cortisol. (G) Motifs for which interactions are induced by all ligands. (H) To explain the tissue-specific actions of CORT125281, Ncor1 and 2 expression was determined in liver, BAT, WAT, quadriceps muscle and hippocampus in vehicle-treated animals. Values are means ± s.e.m. of n = 4–6 mice per group. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0486.

Figure 7

CORT125281 prevents subchronic corticosterone-induced hyperinsulinemia. Mice were exposed to sham or corticosterone (Cort) pellets for 5 days and were treated with CORT125281 (60 mg/kg/day) or mifepristone (MIF, 60 mg/kg/day) via oral gavage. CORT125281 did not prevent corticosterone-induced alterations in body weight (BW) gain (A), fat mass (B), lean mass (C), spleen weight (D), thymus weight (E), total white blood cells (F) (WBC), lymphocyte count (G), monocyte count (H) and eosinophil count (I). CORT125281 partially and mifepristone fully reversed the corticosterone-induced increase in (J) insulin levels, which resulted in improved (K) glucose levels and (L) HOMA-IR. Both CORT125281 and mifepristone seemed to modestly reduce (M) plasma triglycerides, total cholesterol levels (N), but did not affect (O) free fatty acid levels. Values are expressed as means ± s.e.m. of n = 4–6 mice per group. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

CORT125281 does not inhibit subchronic corticosterone-induced GR transcriptional activity. Mice were exposed to vehicle- or corticosterone-pellets for 5 days and were treated with CORT125281 (60 mg/kg/day) or mifepristone (60 mg/kg/day) via oral gavage. CORT125281 did not prevent the corticosterone-induced upregulation of GR target genes in (A) liver, quadriceps muscle (B), brown adipose tissue (C) (BAT), white adipose tissue (D) (WAT) and (E) the hippocampus. CORT125281 additionally did not affect expression of insulin target genes G6p and Pepck in liver, Glut4 and Lpl in WAT and Glut4 and Gsk3b in muscle. Values are means ± s.e.m. of n = 4–6 mice per group. Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.