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. 2019 Oct 1;160(10):2215-2229.
doi: 10.1210/en.2019-00357.

Sex Differences in Adrenal Bmal1 Deletion-Induced Augmentation of Glucocorticoid Responses to Stress and ACTH in Mice

William C Engeland  1 , Logan Massman  1 , Lauren Miller  1 , Sining Leng  2 , Emanuele Pignatti  2 , Lorena Pantano  3 , Diana L Carlone  2   4 , Paulo Kofuji  1 , David T Breault  2   4
Affiliations

Sex Differences in Adrenal Bmal1 Deletion-Induced Augmentation of Glucocorticoid Responses to Stress and ACTH in Mice

William C Engeland et al. Endocrinology. .

Abstract

The circadian glucocorticoid (GC) rhythm is dependent on a molecular clock in the suprachiasmatic nucleus (SCN) and an adrenal clock that is synchronized by the SCN. To determine whether the adrenal clock modulates GC responses to stress, experiments used female and male Cyp11A1Cre/+::Bmal1Fl/Fl knockout [side-chain cleavage (SCC)-KO] mice, in which the core clock gene, Bmal1, is deleted in all steroidogenic tissues, including the adrenal cortex. Following restraint stress, female and male SCC-KO mice demonstrate augmented plasma corticosterone but not plasma ACTH. In contrast, following submaximal scruff stress, plasma corticosterone was elevated only in female SCC-KO mice. Adrenal sensitivity to ACTH was measured in vitro using acutely dispersed adrenocortical cells. Maximal corticosterone responses to ACTH were elevated in cells from female KO mice without affecting the EC50 response. Neither the maximum nor the EC50 response to ACTH was affected in male cells, indicating that female SCC-KO mice show a stronger adrenal phenotype. Parallel experiments were conducted using female Cyp11B2 (Aldosterone Synthase)Cre/+::Bmal1Fl/Fl mice and adrenal cortex-specific Bmal1-null (Ad-KO) mice. Plasma corticosterone was increased in Ad-KO mice following restraint or scruff stress, and in vitro responses to ACTH were elevated in adrenal cells from Ad-KO mice, replicating data from female SCC-KO mice. Gene analysis showed increased expression of adrenal genes in female SCC-KO mice involved in cell cycle control, cell adhesion-extracellular matrix interaction, and ligand receptor activity that could promote steroid production. These observations underscore a role for adrenal Bmal1 as an attenuator of steroid secretion that is most prominent in female mice.

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Conflict of interest statement

Disclosure Summary: The authors have nothing to disclose.

All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.

Figures

Figure 1.
Figure 1.
Adrenal BMAL1 and mPER2Luc rhythms in female and male Cyp11A1Cre/+::Bmal1FL/FL KO mice. In Cyp11A1Cre/+::Bmal1+/+ CTRL mice, nuclear BMAL1 labeling is observed in the adrenal cortex (zona glomerulosa and zona fasciculata) and medulla of (A) female and (C) male mice. In (B) female and (D) male KO mice, nuclear BMAL1 labeling is absent in the adrenal cortex but not the medulla. Mice were 2 to 3 mo of age. (A–D) BMAL1 immunofluorescence labeling; med., medulla; zF, zona fasciculata; zG, zona glomerulosa. Border between cortex and medulla denoted by dashed yellow lines. Scale bars, 50 µm. Cyp11A1Cre/+::Bmal1Fl/Fl mice were intercrossed with mPER2Luc mice and adrenal mPER2Luc was monitored ex vivo to assess the mPER2 clock protein rhythm (9). (E) Adrenals from a CTRL female Cyp11A1Cre/+::Bmal1+/+::PER2Luc (Cre-CTRL) mouse show mPER2Luc rhythms that persist for ∼3 to 4 d ex vivo, whereas adrenals from a female Cyp11A1Cre/+::Bmal1Fl/Fl::PER2Luc KO mouse show reduced mPER2Luc rhythmicity. (F) Peak amplitude dampened ex vivo over daily cycles in CTRL mice, but most adrenals from female KO mice (seven of eight) showed no peaks. (G) Adrenals from a CTRL male (Cre-CTRL) mouse show a mPER2Luc rhythm for 4 to 5 d, whereas adrenals from a male KO mouse show a lower amplitude rhythm. Peak amplitudes were lower in (F) female vs (H) male CTRL adrenals. (H) Amplitudes in daily cycles of mPER2Luc of male KO adrenals were decreased compared with male CTRL adrenals (mean ± SEM; n = 8 to 13 mice; *P < 0.05). Adrenal Bmal1 deletion results in parallel reduction in the amplitude of the mPER2Luc rhythm in female and male mice.
Figure 2.
Figure 2.
Comparison of in vivo plasma corticosterone responses to acute stress and in vitro adrenal cell corticosterone responses to ACTH in male and female CypA1Cre/+::Bmal1+/+ (Cre-CTRL) and CypA1+/+::Bmal1FL/FL (Bmal-CTRL) mice. Prestress plasma corticosterone in the morning did not differ between (A) female or (B) male Cre-CTRL and Bmal1-CTRL mice, but responses to 15-min restraint stress were reduced in (A) female and (B) male Cre-CTRL mice. Corticosterone secretion from adrenal cells in vitro showed the same maximum but higher EC50 responses to ACTH in (C) female and male (D) Cre-CTRL mice. Mean ± SEM; n = 4 to 8 mice; *P < 0.05.
Figure 3.
Figure 3.
Augmented corticosterone responses to acute stress in male and female adrenal CypA1Cre/+::Bmal1Fl/Fl KO mice. Prestress plasma corticosterone in (A) female and (B) male mice was not different between CypA1Cre/+::Bmal1+/+ (Cre-CTRL) and CypA1Cre/+::Bmal1FL/FL KO mice. In response to tail-clip sampling and 15-min restraint, plasma corticosterone responses were augmented in (A) female and (B) male KO mice compared with Cre-CTRL mice. Plasma ACTH was increased in (C) female and (D) male Cre-CTRL mice compared with KO mice. The ratio of plasma corticosterone (compound B) to the log plasma ACTH, an indirect assessment of adrenal responsiveness to ACTH, was increased in (E) female and (F) male KO mice compared with Cre-CTRL mice. Mean ± SEM; n = 5 to 17 mice; *P < 0.05.
Figure 4.
Figure 4.
Augmented corticosterone responses to submaximal scruff stress in female but not male adrenal CypA1Cre/+::BmalFl/Fl KO mice. Plasma corticosterone at 15 min after brief (10-s) scruff stress was increased in (A) female KO mice compared with CTRL mice, but not in (B) male KO mice compared with Cre-CTRL mice. (C and D) Plasma ACTH following scruff stress was not different between (C) female or (D) male KO mice compared with CTRL mice. The ratio of plasma corticosterone (compound B) to the log plasma ACTH was increased in (E) female but not (F) male KO mice compared with Cre-CTRL mice. Mean ± SEM; n = 5 to 9 mice; *P < 0.05.
Figure 5.
Figure 5.
Corticosterone responses to ACTH in acutely dispersed adrenal cells from female and male CypA1Cre/+::BmalFl/Fl KO mice. (A) Corticosterone responses to ACTH were increased in adrenal cells from female KO mice compared with Cre-CTRL mice. (B) In contrast, corticosterone responses to ACTH of adrenal cells from male KO mice were not different from Cre-CTRL mice. Mean ± SEM; n = 4 to 14 mice; *P < 0.05.
Figure 6.
Figure 6.
Augmented corticosterone responses in female adrenal ASCre/+::Bmal1Fl/Fl KO mice to acute stress in vivo and to ACTH in vitro. (A) Prestress plasma corticosterone was not different between female CTRL and KO mice. In response to tail-clip sampling and 15-min restraint, plasma corticosterone was augmented in female ASCre/+::Bmal1Fl/Fl KO mice compared with ASCre/+::Bmal1+/+ CTRL mice. (B) Plasma corticosterone at 15 min after brief (10-s) scruff stress also was increased in female KO mice compared with CTRL mice. *P < 0.05. (C) Acutely dispersed adrenal cells from female ASCre/+::Bmal1FL/FL KO mice secreted increased corticosterone to ACTH in vitro compared with female CTRL mice. Mean ± SEM; n = 3 to 5 mice; *P < 0.05.
Figure 7.
Figure 7.
RNA-seq of CypA1Cre/+::Bmal1Fl/Fl KO and CypA1Cre/+::Bmal1+/+ Cre-CTRL adrenals reveals sex-biased gene expression. (A) RNA-seq analysis revealed 242 genes in female adrenals (157 upregulated and 85 downregulated) and (B) 192 genes in male adrenals (106 upregulated and 86 downregulated) significantly changed, with a false discovery rate <0.05 and fold change >1.5 (upregulated) or <0.5 (downregulated) (n = 4 to 6). Heat maps of reads per kilobase million values from (A) female and (B) male CypA1Cre/+::Bmal1fl/fl KO and Cre-CTRL adrenals. Dendrograms represent hierarchical clustering of genes (left) and samples (top). (C) KEGG pathways enriched in upregulated (red) or downregulated (blue) genes in female and male data sets. Genes enriched in specific KEGG pathways are listed, with red font indicating uniquely upregulated genes in female KO adrenals.
Figure 8.
Figure 8.
Validation of the RNA-seq analysis. (A) Selected genes that emerged from the RNA-seq data set as differentially expressed either in female or male CypA1Cre/+::Bmal1Fl/Fl KO adrenals were validated by quantitative PCR using independent samples. (B) Star transcripts were not affected by loss of Bmal in female adrenals. Mean ± SEM; n = 5 to 6 mice; *P < 0.05; **P < 0.01; ***P < 0.001.
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