Mouse Adrenal Cortex Research Articles

The ex vivo perfused mouse adrenal gland—a new model to study aldosterone secretion

Aldosterone is a steroid hormone that is important for maintaining the volume and ionic composition of extracellular fluids and is produced in the zona glomerulosa of the adrenal cortex. The basic mechanisms controlling aldosterone secretion are known. However, more detailed studies on the regulation of aldosterone secretion often fail due to the lack of suitable models: although secretion can be studied in cultured adrenocortical cells under defined conditions, the differentiation status of the cells is difficult to control and the complex anatomy of the adrenal cortex is lost. In living animals, the physiological context is intact, but the influences are manifold and the examination conditions cannot be sufficiently controlled. One method that closes the gap between cell models and studies in living animals is the isolated perfused adrenal gland. In the past, this method has provided important data on the pathophysiology of adrenal glands from larger animals, but the technique was not used in mice. Here, we developed a method for isolation and perfusion of the mouse adrenal gland to study aldosterone secretion. This technique preserves the complex anatomical and functional context of the mouse adrenal cortex, to ensure defined experimental conditions and to minimize extra-adrenal influences. Initial series of experiments with the ex vivo perfused mouse adrenal gland show that this model offers the possibility for unique insights into pathophysiological regulatory principles and is suitable for the use of genetically modified mouse models.

Thromboxane A2 Modulates de novo Synthesis of Adrenal Corticosterone in Mice via p38/14-3-3γ/StAR Signaling.

Prostanoids are endogenous lipid bioactive mediators that play essential roles in physiological processes such as glucocorticoid secretion. Here, it is found that the thromboxane (Tx)A2 receptor (TP) is highly expressed in the adrenal cortex of mice. Both global and adrenocortical-specific deletion of the TP receptor lead to increased adiposity in mice by elevating corticosterone synthesis. Mechanistically, the TP receptor deletion increases the phosphorylation of steroidogenic acute regulatory protein (StAR) and corticosterone synthesis in adrenal cortical cells by suppressing p-p38-mediated phosphorylation of 14-3-3γ adapter protein at S71. The activation of the p38 in the adrenal cortical cells by forced expression of the MKK6EE gene attenuates hypercortisolism in TP-deficient mice. These observations suggest that the TxA2/TP signaling regulates adrenal corticosterone homeostasis independent of the hypothalamic-pituitary-adrenal axis and the TP receptor may serve as a promising therapeutic target for hypercortisolism.

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis.

Cholesterol is the precursor of all steroids, but how cholesterol flux is controlled in steroidogenic tissues is poorly understood. The cholesterol exporter ABCG1 is an essential component of the reverse cholesterol pathway and its global inactivation results in neutral lipid redistribution to tissue macrophages. The function of ABCG1 in steroidogenic tissues, however, has not been explored. To model this, we inactivated Abcg1 in the mouse adrenal cortex, which led to an adrenal-specific increase in transcripts involved in cholesterol uptake and de novo synthesis. Abcg1 inactivation did not affect adrenal cholesterol content, zonation, or serum lipid profile. Instead, we observed a moderate increase in corticosterone production that was not recapitulated by the inactivation of the functionally similar cholesterol exporter Abca1. Altogether, our data imply that Abcg1 controls cholesterol uptake and biosynthesis and regulates glucocorticoid production in the adrenal cortex, introducing the possibility that ABCG1 variants may account for physiological or subclinical variation in stress response.

OR02-06 FGFR2 Mediates Zona Glomerulosa Morphogenesis, Aldosterone Secretion And Β-Catenin-induced Adrenal Hyperplasia

Abstract Disclosure: V. Chortis: None. K. Dulanjalee: None. N.A. Guagliardo: None. M.S. Shah: None. R. Claudio: None. P.Q. Barrett: None. K.S. Borges: None. D.L. Carlone: None. D.T. Breault: None. Fibroblast Growth Factor Receptors (FGFRs) are tyrosine kinase receptors with critical roles in organogenesis and tissue homeostasis. We have recently shown that Fgfr2 deletion in the outermost zone of the mouse adrenal cortex (zona Glomerulosa, zG) during post-natal development leads to disordered morphology. Interestingly, Fgfr2 is up-regulated in the zG of mice with adrenal hyperplasia due to constitutive activation of Wnt/beta-catenin signaling. Here, we seek to delineate the role of FGFR2 in adrenal morphology and function during postnatal development, adulthood homeostasis, as well as during β-catenin-induced adrenal hyperplasia. To address this, we used i) a mouse model of conditional Fgfr2 loss (Fgfr2-cKO), in which zG-specific Fgfr2 deletion begins during early post-natal development, and ii) an inducible mouse model (Fgfr2-icKO), in which Fgfr2 deletion in the zG during adult life is tamoxifen-dependent. Fgfr2 loss in Fgfr2-cKO mice disrupted zG cell differentiation in steroidogenically mature cells and limited their capacity to proliferate and undergo trans-differentiation into the adjacent zona Fasciculata. zG cell de-differentiation was also evident upon acute loss of Fgfr2 in adult Fgfr2-icKO mice. Furthermore, Fgfr2 loss in a mouse model of beta-catenin-induced zG hyperplasia was sufficient to fully abrogate adrenal hyperplasia. Treatment with pan-FGFR small molecule inhibitors suppressed cellular proliferation in the mouse zG and inhibited aldosterone secretion. Taken together, these results suggest FGFR2 has a critical role in normal adrenal development and tissue homeostasis and is an essential mediator of beta-catenin-induced adrenal hyperplasia. FGFR2 targeting may be meaningful in patients with conditions associated with aldosterone excess. Presentation: Thursday, June 15, 2023

Characterizing and Quenching Autofluorescence in Fixed Mouse Adrenal Cortex Tissue.

Tissue autofluorescence of fixed tissue sections is a major concern of fluorescence microscopy. The adrenal cortex emits intense intrinsic fluorescence that interferes with signals from fluorescent labels, resulting in poor-quality images and complicating data analysis. We used confocal scanning laser microscopy imaging and lambda scanning to characterize the mouse adrenal cortex autofluorescence. We evaluated the efficacy of tissue treatment methods in reducing the intensity of the observed autofluorescence, such as trypan blue, copper sulfate, ammonia/ethanol, Sudan Black B, TrueVIEWTM Autofluorescence Quenching Kit, MaxBlockTM Autofluorescence Reducing Reagent Kit, and TrueBlackTM Lipofuscin Autofluorescence Quencher. Quantitative analysis demonstrated autofluorescence reduction by 12-95%, depending on the tissue treatment method and excitation wavelength. TrueBlackTM Lipofuscin Autofluorescence Quencher and MaxBlockTM Autofluorescence Reducing Reagent Kit were the most effective treatments, reducing the autofluorescence intensity by 89-93% and 90-95%, respectively. The treatment with TrueBlackTM Lipofuscin Autofluorescence Quencher preserved the specific fluorescence signals and tissue integrity, allowing reliable detection of fluorescent labels in the adrenal cortex tissue. This study demonstrates a feasible, easy-to-perform, and cost-effective method to quench tissue autofluorescence and improve the signal-to-noise ratio in adrenal tissue sections for fluorescence microscopy.

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex.

Steroid hormones are synthesized through enzymatic reactions using cholesterol as the substrate. In steroidogenic cells, the required cholesterol for steroidogenesis can be obtained from blood circulation or synthesized de novo from acetate. One of the key enzymes that control cholesterol synthesis is 24-dehydrocholesterol reductase (encoded by DHCR24). In humans and rats, DHCR24 is highly expressed in the adrenal gland, especially in the zona fasciculata. We recently reported that DHCR24 was expressed in the mouse adrenal gland's inner cortex and also found that thyroid hormone treatment significantly upregulated the expression of Dhcr24 in the mouse adrenal gland. In the present study, we showed the cellular expression of DHCR24 in mouse adrenal glands in early postnatal stages. We found that the expression pattern of DHCR24 was similar to the X-zone marker gene 20αHSD in most developmental stages. This finding indicates that most steroidogenic adrenocortical cells in the mouse adrenal gland do not synthesize cholesterol locally. Unlike the 20αHSD-positive X-zone regresses during pregnancy, some DHCR24-positive cells remain present in parous females. Conditional knockout mice showed that the removal of Dhcr24 in steroidogenic cells did not affect the overall development of the adrenal gland or the secretion of corticosterone under acute stress. Whether DHCR24 plays a role in conditions where a continuous high amount of corticosterone production is needed requires further investigation.

RF33 | PSAT79 Chronic Activation of Gq-Signaling in the Mouse Adrenal Cortex Disrupts Zonation and Causes Aldosterone Production in the Zona Fasciculata

Abstract Background The mammalian adrenal cortex is composed of the zona glomerulosa (ZG), zona fasciculata (ZF), and in some species the zona reticularis (ZR). Each layer is characterized by a specific steroidogenic enzyme expression pattern that leads to selective production of mineralocorticoids, glucocorticoids or adrenal androgens. ZG-specific expression of cytochrome P450 aldosterone synthase (Cyp11b2) and aldosterone biosynthesis are renin-angiotensin-dependent processes in which the Gq-signaling pathway is activated by angiotensin II (AngII). Gq-signaling can be exogenously activated using Gq-coupled designer receptors exclusively activated by designer drug (GqDREADD) technology. Using transgenic mice with targeted expression of GqDREADD to the adrenal cortex, we previously demonstrated that short-term receptor activation increased adrenal Cyp11b2 and aldosterone production. Herein, we hypothesized that prolonged activation of mouse adrenal cortex Gq-signaling would exacerbate the aldosterone levels and promote the transdifferentiation of ZF cells to ZG cells. Methods Transgenic AS+/Cre hM3Dq (GqDREADD) female mice were engineered through crossing GqDREADD mice with Cyp11b2-regulated Cre recombinase mice. Mice were placed on a high sodium diet (to suppress physiologic renin) and orally provided the GqDREADD ligand, clozapine N-oxide (CNO; n=10) or control (n=8) for 6 weeks followed by collection of blood and adrenals. Mineralocorticoid levels were quantified using LC-MS/MS. Adrenal RNA was isolated and assessed by RNAseq for transcriptomic changes, and RT-qPCR analysis for ZG- and ZF-specific transcript markers, Cyp11b2 and Cyp11b1 (marker of ZF), respectively. Transcripts for β-catenin (Ctnnb1) and axin-2 (Axin2) (markers of the WNT signaling pathway) were also analyzed. Immunofluorescence (IF) studies examined adrenal expression of Cyp11b1 and Cyp11b2 in mice treated for 1 month. Results CNO treatment caused plasma aldosterone to increase 613-fold from 7.9 ±2.5 pg/mL to 4810.7 ±1110.7 pg/mL (p<0.01). Levels of 18-hydroxycorticosterone increased 70-fold from 144.9 ±22.0 to 10143.2 ±1479.6 pg/mL (p<0.0001). Corticosterone levels were unaffected. RT-qPCR analysis showed adrenal Cyp11b2 transcripts to be 224-fold higher (p<0.0001) in the CNO-treated group while a 30% decrease in Cyp11b1 levels was observed (p<0.01) compared to the controls. IF demonstrated hybrid ZF cells aberrantly expressing both Cyp11b2 and Cyp11b1. Within the normal mouse adrenal, the outer ZG exhibits high WNT/β-catenin (Ctnnb1) activation. To determine if chronic CNO treatment impacted overall adrenal WNT signaling, Ctnnb1 and Axin2 (a downstream β-catenin gene target) were examined. Analysis revealed no statistical difference in Ctnnb1 and Axin2 transcript levels in treated mice. RNAseq revealed that 242 transcripts were increased in adrenals from treated mice, while 109 decreased (>3-fold change; p<0.01). Induced transcripts include Cyp11b2 and its transcriptional regulators, Nr4a1 and Nr4a2. Conclusion Overall, these findings demonstrate that chronic Gq-signaling is sufficient to cause ZF cells to produce aldosterone and express Cyp11b2. The findings also suggest that the adrenocortical WNT signaling pathway is not increased by the activation of Gq-signaling. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:36 p.m. - 12:41 p.m.

Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development.

Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.

Effect of Inactivation of Mst1 and Mst2 in the Mouse Adrenal Cortex.

Recent conditional knockout of core components of the Hippo signaling pathway in the adrenal gland of mice has demonstrated that this pathway must be tightly regulated to ensure proper development and maintenance of the adrenal cortex. We report herein that the most upstream kinases of the pathway, the mammalian STE20-like protein kinases 1 and 2 (MST1and MST2, respectively), are expressed in the mouse adrenal cortex with MST2 expression being restricted to the zona glomerulosa (zG). To further explore the role of Hippo signaling in adrenocortical cells, we conditionally deleted Mst1/2 in steroidogenic cells using an Nr5a1-cre strain (Mst1 flox/flox ; Mst2 flox/flox ; Nr5a1-cre). Our results show that the loss of MST1/2 leads to the premature and progressive accumulation of subcapsular GATA4+, WT1+ adrenal gonadal primordium (AGP)-like progenitor cells starting at 2 months of age without affecting aldosterone and corticosterone secretion. To help us understand this phenotype, microarray analyses were performed on adrenal glands from 2-month-old mutant and control mice. Gene expression analyses revealed that loss of Mst1/2 leads to the overexpression of known downstream target genes (Ajuba, Aqp1, Fn1, Ibsp, Igf1, Igfbp2, Mmp2, Thbs1) of the main effector of Hippo signaling, YAP; and underexpression of genes (Agtr1b, Ecgr4, Hsd3b6, Nr0b1, Tesc, Vsnl1) that are normally specifically expressed in the zG or overexpressed in the zG compared to the zona fasciculata (zF). Together, these results suggest that MST1/2 regulates Hippo signaling activity in the adrenal cortex and that these two kinases are also involved in the fine tuning of zG cell function or differentiation.

Androgen Receptor Is Dispensable for X-Zone Regression in the Female Adrenal but Regulates Post-Partum Corticosterone Levels and Protects Cortex Integrity.

Adrenal androgens are fundamental mediators of ovarian folliculogenesis, embryonic implantation, and breast development. Although adrenal androgen function in target tissues are well characterized, there is little research covering the role of androgen-signaling within the adrenal itself. Adrenal glands express AR which is essential for the regression of the X-zone in male mice. Female mice also undergo X-zone regression during their first pregnancy, however whether this is also controlled by AR signaling is unknown. To understand the role of the androgen receptor (AR) in the female adrenal, we utilized a Cyp11a1-Cre to specifically ablate AR from the mouse adrenal cortex. Results show that AR-signaling is dispensable for adrenal gland development in females, and for X-zone regression during pregnancy, but is required to suppress elevation of corticosterone levels post-partum. Additionally, following disruption to adrenal AR, aberrant spindle cell development is observed in young adult females. These results demonstrate sexually dimorphic regulation of the adrenal X-zone by AR and point to dysfunctional adrenal androgen signaling as a possible mechanism in the early development of adrenal spindle cell hyperplasia.

HOX genes promote cell proliferation and are potential therapeutic targets in adrenocortical tumours

BackgroundUnderstanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment.MethodsWe used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options.ResultsOur human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor.ConclusionsThese studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

SUN-LB42 The Sexually Dimorphic Response of the Mouse Adrenal Inner Cortex to Thyroid Hormone Treatment

The gender bias in adrenal diseases has been noticed for a long time. Mouse studies have shown that the adrenal gland is sexually dimorphic at different levels, such as transcriptome, histology, and cell renewal. However, the mechanism behind this sexual dimorphism is not fully understood. Here, we used RNA-seq to demonstrate how male and female adrenals respond differently to the same external cue, the thyroid hormone (T3) treatment, which directly elicits its function on the adrenal inner cortex by changing the cell fate of this population. Through the comparison of the adrenal gland transcriptomes from males and females with T3 or saline treatment, we found that more genes in female adrenals were responsive to the T3 treatment, whereas the fold change of the gene expressions was greater in male adrenals. Statistical analysis identified 104 sexually dimorphic T3-responsive genes. Immunostaining results showed that many of these genes were expressed in the adrenal gland inner cortex, which contains a unique cell population called X-zone (20-alpha-HSD positive). Previous studies showed that T3 treatment leads to the expansion of the 20αHSD-positive zone both in males and in females. Here we found that the top sexually dimorphic T3-responsive gene was expressed in the adrenal inner cortex partially colocalized with X-zone. Under T3 treatment, this unique cell population that surrounds the 20-alpha-HSD positive X-zone became obvious only in females but not in males. Our findings not only identified several novel marker genes for the adrenal inner cortex but also highlighted the sex-specific response of thyroid hormone action in the mouse adrenal gland.

SUN-LB43 DLK1 Expressing Cells Contribute to the Zonation of the Adrenal Gland

The adrenal cortex is a dynamic organ that undergoes self-renewal and remodeling in response to the demand for steroids. In the mouse it is divided into two concentric layers, the outer zona glomerulosa and the inner zona fasciculata (ZF), that secrete aldosterone and corticosterone, respectively. Cell fate mapping studies have shown that the maintenance of the cortex relies on a pool of stem/progenitor cells located in the capsular and subcapsular compartments. Two interconnected cell populations have been identified, subcapsular undifferentiated cells secreting the morphogen Sonic Hedgehog (Shh) and capsular Gli1+ cells, which can transduce the Shh signal (1); both populations are precursors of steroidogenic cells and newly formed cells migrate in a centripetal fashion to repopulate the gland until they reach the juxtamedullary region where they undergo senescence and apoptosis. Moreover, our lab has shown that the Notch atypical ligand Delta-Like homologue 1 (Dlk1) is expressed in partially undifferentiated cells of the subcapsular region in rat (2)and human (3)adrenals, whilst it is mostly expressed in capsular cells in mice (4,5). To investigate whether Dlk1 expressing cells contribute to the zonation of the adrenal cortex we conducted lineage tracing analyses using a tamoxifen inducible Dlk1CreERT2mouse model carrying the R26tdTom reporter. Pregnant dames were injected with tamoxifen at embryonic day (e) 12.5 and pups were culled at postnatal day (p) 10 and p38. Analysis of tdTomato expression showed that 35% (p10) and 24% (p38) of Steroidogenic Factor 1(Sf1)+cortical cells were tdTomato+, revealing that capsular Dlk1+cells are steroidogenic precursors. On the other hand, postnatal tamoxifen injections (p0) showed tdTomato+/Sf1+ cells only in 1-2% in cortical cells after 24-months chase, suggesting that the contribution of Dlk1+cells to adrenocortical self-renewal is limited postnatally.However, the Dlk1+population could be reactivated in the adult mouse treated with dexamethasone and was shown to contribute to the regeneration of the ZF once dexamethasone treatment was ceased. 1. King P, et al. Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages. Proc Natl Acad Sci(2009) 106:21185-211902. Guasti L, et al. Dlk1 Up-Regulates Gli1 Expression in Male Rat Adrenal Capsule Cells Through the Activation of β1 Integrin and ERK1/2. Endocrinology(2013) 154:4675-46843. Hadjidemetriou I, et al. DLK1/PREF1 marks a novel cell population in the human adrenal cortex. J Steroid Biochem Mol Biol(2019) 193:1054224. Guasti L, Candy Sze WC, McKay T, Grose R, King PJ. FGF signalling through Fgfr2 isoform IIIb regulates adrenal cortex development. Mol Cell Endocrinol(2013) 371:182-1885. Heikkilä M, et al. Wnt-4 Deficiency Alters Mouse Adrenal Cortex Function, Reducing Aldosterone Production. Endocrinology(2002) 143:4358-4365

Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrançois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

Sleeping through anything: The effects of unpredictable disruptions on mouse sleep, healing, and affect.

Many aspects of the laboratory environment are not tailored to the needs of rodents, which may cause stress. Unpredictable stressors can cause ulcers, prolonged pituitary-adrenal activation, and anhedonia. Similarly, pain has been demonstrated to slow wound healing, and mice experiencing pain exhibit altered behavior. However it is unknown how husbandry, which occurs when the mice are inactive, and lack of analgesia, specifically in a punch biopsy procedure, effects animal physiology, behavior, and welfare, particularly as it relates to sleep fragmentation. We hypothesized that sleep fragmentation, induced by unpredictable husbandry and lack of pain management will slow wound healing. Two main treatments were tested in a factorial design in C57BL/6 mice of both sexes (64 mice total); 1) analgesia (carprofen and saline) and 2) sleep disruptions (random and predictable). Mice were singly housed in a non-invasive sleep monitoring apparatus on arrival (Day -4). Disruption treatments were applied from Day -3 to 2. All mice received a punch biopsy surgery (Day 0) with topical lidocaine gel and their analgesic treatment prior to recovery, and on Days 1 and 2. Nesting behavior was assessed daily and a sugar cereal consumption test, as a measure of anhedonia, was conducted on Days -1 to 2. On Day 3, mice were euthanized and wound tissue and adrenal glands were collected. We found that the disruption predictability had no effect on mouse sleep, wound healing, or adrenal cortex:medulla ratio. It’s possible that the disruption period was not long enough to induce chronic stress. However, male mice who received analgesia slept more than their female counterparts; this may be related to sex differences in pain perception. Overall, it does not appear that the predictability of disturbance effects sleep fragmentation or stress responses, indicating that husbandry activities do not need to occur at set predictable times to improve welfare.

Dissociation of Molecular and Endocrine Circadian Rhythms in Male Mice Lacking Bmal1 in the Adrenal Cortex.

The circadian rhythm of glucocorticoids affects diverse physiological systems, including stress responses and the coordination of rhythmic functions in peripheral and central tissues. Circadian clocks are considered to be important coordinators of glucocorticoid release and loss of the core clock component Brain and muscle Arnt-like protein-1 leads to ablation of behavioral and physiological rhythms, hypocortisolism, impaired ACTH, and behavioral stress responses. Transplantation and conditional clock gene knock-down studies in mice suggest an important role of local adrenocortical clock function in this context. Here, we present a Cre-loxP-mediated conditional knockout of Bmal1 in the steroidogenic cells of the adrenal cortex in mice. Mutant animals show a loss of molecular clock gene activity rhythms in this tissue with subsequent disruption of rhythmic steroidogenic gene expression. However, despite this loss of normal clock rhythmicity in the adrenal cortex, behavioral and physiological rhythms and acute stress responses persist in mutant mice. These findings reveal a dissociation of transcriptional and endocrine rhythm regulation in the adrenal cortex, arguing for a less pivotal function of the local clock machinery in the regulation of circadian and acute glucocorticoid outputs.

The Inwardly Rectifying Potassium Channel GIRK4 Plays a Role in Aldosterone Production in The Mouse Adrenal Cortex

ObjectiveDetermine the role of wild‐type GIRK4 in the mouse adrenal in relation to Aldosterone regulation.Aldosterone plays a key role in salt homeostasis and blood pressure, its dysregulation accounts for up to 10% of hypertension. Significant portions of these cases are caused by aldosterone producing adenomas (APAs) of the adrenal cortex, and have been shown to harbour mutations in the inwardly rectifying potassium channel GIRK4. Expression of GIRK4 has since been shown in the aldosterone producing zona glomerulosa (ZG) cells of the adrenal cortex. Although the role of these mutant GIRK4 channels have been well studied in vitro, little is known about the role of wild‐type GIRK4 in vivo. Here we utilize the GIRK4 −/− mouse to investigate the role of GIRK4 in aldosterone regulation and blood pressure.Typically aldosterone secretion from the ZG cells of the adrenal cortex is mediated by depolarization upon stimulation with angiotensin II. The presence of GIRK4 in ZG cells is thought to help maintain a hyperpolarized membrane potential post stimulation, and reduce spontaneous depolarization, thereby preventing autonomous aldosterone production.Paradoxically our pilot data has shown a decrease in CYP11B2 gene expression (1.82 fold, P<0.05) and plasma aldosterone levels (1.63 fold, P<0.05) in GIRK4 −/− mice compared to wild‐type animals (Figure). This suggests cells lacking GIRK4 maintain a hyperpolarized membrane potential, and hence GIRK4 is not solely responsible for hyperpolarization of the ZG cells of the adrenal cortex.In order to elucidate the molecular basis for this observation, we are conducting RNAseq analysis of adrenals from wild‐type and GIRK4 −/− mice. This will reveal transcriptomic changes that may explain the observed decrease in aldosterone production, as well as other potential pathways involved in aldosterone regulation in the adrenal cortex.Support or Funding InformationThis work was supported by the British Heart Foundation (PhD studentship to IH FS 12/78/29875)

Fgfr2 is required for the expansion of the early adrenocortical primordium

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from intermediate mesoderm of the anterior urogenital ridge. Previous work has pinpointed a minor role of the FGFR2IIIb isoform in expansion and differentiation of the fetal adrenal cortex in mice but did not address the complete role of FGFR2 and FGFR1 signaling in adrenocortical development. Here, we show that a Tbx18cre line mediates specific recombination in the coelomic epithelium of the anterior urogenital ridge which gives rise by a delamination process to the adrenocortical primordium. Mice with conditional (Tbx18cre-mediated) deletion of all isoforms of Fgfr2 exhibited severely hypoplastic adrenal glands around birth. Cortical cells were dramatically reduced in number but showed steroidogenic differentiation and zonation. Neuroendocrine chromaffin cells were also reduced and formed a cell cluster adjacent to but not encapsulated by steroidogenic cells. Analysis of earlier time points revealed that the adrenocortical primordium was established in the intermediate mesoderm at E10.5 but that it failed to expand at subsequent stages. Our further experiments show that FGFR2 signaling acts as early as E11.5 to prevent apoptosis and enhance proliferation in adrenocortical progenitor cells. FGFR1 signaling does not contribute to early adrenocortical development. Our work suggests that FGFR2IIIb and IIIc isoforms largely act redundantly to promote expansion of the adrenocortical primordium.

Phosphodiesterase 8B and cyclic AMP signaling in the adrenal cortex.

Bilateral adrenocortical hyperplasia (BAH) in humans and mice has been recently linked to phosphodiesterase (PDE) 8B (PDE8B) and 11 (PDE11A) defects. These findings have followed the discovery that defects of primary genes of the cyclic monophosphatase (cAMP) signaling pathway, such as guanine nucleotide binding alpha subunit and PRKAR1A, are involved in the pathogenesis of BAH in humans; complete absence of Prkar1a in the adrenal cortex of mice also led to pathology that mimicked the human disease. Here, we review the most recent findings in human and mouse studies on PDE8B, a cAMP-specific PDE that appears to be highly expressed in the adrenal cortex and whose deficiency may underlie predisposition to BAH and possibly other human diseases.

Changes in morphology and function of adrenal cortex in mice fed a high-fat diet.

Obesity is a major risk factor for the development of type 2 diabetes and other debilitating diseases. Obesity and diabetes are intimately linked with altered levels of adrenal steroids. Elevated levels of these hormones induce insulin resistance and cause cardiovascular diseases. The mechanisms underlying obesity-related alterations in adrenal steroids are still not well understood. Here, we investigated how diet-induced obesity affects the morphology and function of the mouse adrenal cortex. We fed animals either a high-fat diet (HFD) or a normal diet (60% kcal from fat or 10% kcal from fat, respectively) for 18 weeks. We then assessed various aspects of adrenal gland morphology and function, as well as basal plasma concentrations of steroid hormones and ACTH. We show that adrenal glands of mice fed a HFD release more corticosterone and aldosterone, resulting in higher plasma levels. This increase is driven by adrenal cortical hyperplasia, and by increased expression of multiple genes involved in steroidogenesis. We demonstrate that diet-induced obesity elevates Sonic hedgehog signaling in Gli1-positive progenitors, which populate the adrenal capsule and give rise to the steroidogenic cells of the adrenal cortex. Feeding animals with a HFD depletes Gli1-positive progenitors, as the adrenal cortex expands. This work provides insight into how diet-induced obesity changes the biology of the adrenal gland. The association of these changes with increased Shh signaling suggests possible therapeutic strategies for obesity-related steroid hormone dysfunction.