Adrenal Expression Research Articles

12544 Notch4 And Gbp7 Genetic Contribution To Primary Aldosteronism Pathology

Abstract Disclosure: N.S. Mohd Rizam: None. F.A. Pauzi: None. M. Choy: None. S. Williams: None. A. Aminuddin: None. F. Abdul Latif: None. S. Syed Mohammed Nazri: None. M. Mustangin: None. G. Tan: None. X. Wu: None. E. Ng: None. K. Laycock: None. N. Sukor: None. A.B. Nasruddin: None. M.J. Brown: None. B. Keavney: None. E.A. Azizan: None. Primary aldosteronism (PA) is a common curable cause of hypertension (5-10% cases) yet markedly underdiagnosed. Heredity contributes 25-64% of inter-individual blood pressure variation. Genome-wide association studies have identified a proportion of these genetic factors, whole-genome sequencing (WGS) could provide deeper genetic resolution. We therefore performed WGS on PA patients to elucidate genetic contributors for aldosterone related hypertension. In PA patients of Malay or Black ethnicity, missense mutations in NOTCH4 were frequently encountered (21 of 100). Four of the seven mutations identified were rare (gnomAD allele frequency = 0) and damaging (CADD score >20). The protein is a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 that regulates cell-fate determination. Whereas in Black PA patients or PA patients with no apparent adenoma, an enrichment of two rare missense mutations in GBP7 occurred compared to the global population (7 of 100). The mutations in GBP7 were verified as the encoded protein hydrolyzes GTP to GMP associated with the aldosterone synthesis pathway. Bulk adrenal expression of NOTCH4 and GBP7 were 11.7 and 1.145 transcript per million (TPM) respectively (data acquired from GTex). Single-cell analysis of human adrenals identified NOTCH4 to be expressed mainly in endothelial cells. Co-culturing of the endothelial cell line (HCMEC) with an adrenocortical cell line (HAC15) increased expression of CYP11B2, encoding the aldosterone synthase enzyme, by 1.82+0.37-fold (n=6, p=0.03). Overexpressing NOTCH4 and GBP7 in HAC15 increased CYP11B2 expression by 1.74+0.08-fold (n=16, p=0.000003) and 1.57+0.21-fold (n=24, p=0.001) respectively. In summary, NOTCH4 and GBP7 may highlight a novel pathway in PA pathology. Presentation: 6/2/2024

Interrogating the estrogen-mediated regulation of adrenocortical Klotho expression using ovariectomized albino rat model exposed to repeated restraint stress.

Reproductive aging is associated with altered stress response and many other menopausal symptoms. Little is known about the adrenal expression of the anti-aging protein Klotho or how it is modulated by estrogen in ovariectomized stressed rats. Fifty-six Wistar female rats were assigned into seven equal groups. Sham-operated (Sham), sham stressed (Sham/STS), ovariectomized (OVR), ovariectomized stressed (OVR/STS), ovariectomized stressed rosiglitazone-treated (OVR/STS/R), ovariectomized stressed estrogen-treated (OVR/STS/E), and ovariectomized stressed estrogen/GW9662 co-treated (OVR/STS/E/GW) groups. All stressed rats were subjected daily to a one-hour restraint stress test for 19days.At the end of the experiment, blood was collected for serum corticosterone (CORT) analysis. Adrenal tissues were obtainedand prepared for polymerase chain reaction (PCR) assay, hematoxylin and eosin (H&E), immunohistochemistry-based identification of Klotho and PPAR-γ, and Oil Red O (ORO) staining. The rise in serum CORT was negligible in the OVR/STS group, in contrast to the Sham/STS group. The limited CORT response in the former group was restored by estrogen and rosiglitazone and blocked by estrogen/GW9226 co-administration. ORO-staining revealed a more evident reduction in the adrenal fat in the OVR/STS group, which was reversed by estrogen and counteracted by GW. Also, there was a comparable expression pattern of Klotho and PPAR-γ in the adrenals. The adrenal Klotho decreased in the OVR/STS group, but was reversed by estrogen treatment. GW9226/estrogen co-treatment interfered with the regulatory effect of estrogen on Klotho. The study suggested modulation of the adrenal Kotho expression by estrogen, in the ovariectomized rats subjected to a restraint stress test. This estrogen-provided adrenal protection might be mediated by PPAR-γ activation.

A mathematical study of the adrenocorticotropic hormone as a regulator of human gene expression in adrenal glands

Abstract In this research, we have introduced compartments for asymptomatic and symptomatic individuals, along with reduced susceptibility, as key factors defining our investigation. The study is carried out in diverse scenarios, considering them as crucial for the essential generation number of the model, set at 3.18( r 0 > 1 {r}_{0}\gt 1 ). The persistent reproduction differential method was used to explore the impact of continuous adrenocorticotropic hormone (ACTH) administration on the global gene expression in primary cultures of both fetal and adult adrenal cells. The study also investigates ACTH’s genetic effects on both adult and fetal human adrenal cells. The conclusion of this study is demonstrated through relevant and correct medical applications.

Chrysin’s dose-dependent effects on steroidogenesis in female BALB/c mice: In vivo study of adrenal, ovarian, and uterine hormone regulation

Background: Chrysin is known for its pharmacological effects and structural resemblance to estrogen. The study explores the impact of chrysin on steroidogenesis focusing on adrenal and ovarian steroidogenic enzymes. Materials and methods: Thirty female BALB/c mice were divided into three groups: a control group and two chrysin-treated groups (50 mg and 100 mg). Gene expression of key steroidogenic enzymes was assessed in adrenal glands and ovaries using RT-qPCR. Uterine expression of estrogen receptor alpha (ERα) was also examined. Histological analysis of adrenal glands and ovaries was performed. Results: High-dose chrysin downregulated CYP17A1 expression in adrenal glands compared to control and low-dose groups. In contrast, 3β-HSD was significantly downregulated in the high-dose group. In ovaries, high dose chrysin reduced aromatase expression. Conclusion: Our findings revealed that chrysin’s impact on steroidogenesis is dose-dependent. By downregulating CYP17A1 in adrenal glands, potentially affecting androgen and estrogen synthesis, and enhancing aromatase expression in ovaries at lower doses.

Quantitative Characterization of Ectopic Adrenal Gene Expression in Fetal Testes in 21-Hydroxylase Deficient Mice.

Testicular adrenal rest tumors (TART) are a frequent and fertility impairing long-term complication in males with classic congenital adrenal hyperplasia. Due to lack of clear experimental data on their origin, they are hypothesized to be derived from ectopic adrenocortical cells within testicular tissue mainly growing upon stimulation by chronically elevated levels of adrenocorticotropin (ACTH). Alternatively, a more totipotent embryological origin has been discussed as the potential source of these tumors. The aim of this study was to quantify alterations of ectopic expression of adrenocortical genes (CYP11B1, CYP11B2, CYP21, MC2R) and the Leydig cell specific marker (INSL3) in testicular tissue of fetal 21-hydroxylase deficient (21OHD) mice. Timed-pregnancy studies were performed using H-2aw18 (aw18)-mice. Testes and adrenals of E15.5 and E18.5 mouse fetuses were used for real-time PCR and immunohistochemistry. Gene expression levels were analyzed for genotype-dependent alterations and compared with immunohistochemistry. While enzymes of steroidogenesis showed a significant increased expression in adrenals of 21OHD mice at both E15.5 and E18.5 compared to wild-type (WT) mice, expression levels were unaltered in testes of 21OHD mice. When compared to WT adrenals a significant increase of INSL3 expression in adrenals of 21OHD mice at E15.5 and E18.5 was detected. Cells with adrenocortical properties in mice fetal testis differ from in situ adrenocortical cells in gene expression and growth at E15.5 and E18.5. These findings suggest that the different local regulation and different local niche in adrenals and testes influence growth of aberrant adrenal cells.

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

Abstract 063: Pregnancy Protects Aged G Protein-coupled Estrogen Receptor 1 Knock-out Mice Against Hypertension

Hypertension, a major cause of premature death worldwide, is more prevalent in postmenopausal women compared to premenopausal women. Data implicate a protective effect of G protein-coupled estrogen receptor 1 (GPER1) in cardiovascular diseases. A recent study showed that previous pregnancy lowers blood pressure in hyperandrogenemic rats. We hypothesized that pregnancy protects against hypertension elicited in aged female mice lacking GPER1. 16-20 month-old GPER1 wild-type (WT) and knock-out (KO) mice with or without a history of former pregnancies were implanted with telemeters for blood pressure recording and placed in metabolic cages for 24h urine collection. Plasma, adrenal glands and kidneys were collected. GPER1 deletion increased mean arterial pressure in virgin mice (125±3 vs. 109±2 mmHg; n=3,7; p=0.0005), and previous pregnancies (PP) eliminated this genotypic effect (106±1 mmHg; n=6; p≤0.0001). Aldosterone plays a critical role in blood pressure regulation, we measured its urinary excretion and revealed lower levels in PP KO mice compared to virgin KO mice (4.8±0.8 vs. 13.0±3.0 pg/day; n=6; p=0.0099). PP KO mice elicited greater levels of circulating estradiol levels compared to virgin KO mice (42.8±5.2 vs. 29.5±3.4 pg/mL; n=8,6; p=0.0451). To explore the role of adrenal estrogen receptors (ER) in regulating aldosterone production, we assessed the adrenal expression of ER and CYP112B, the gene coding for aldosterone synthase. No difference was observed in ERα, however ERβ expression was higher in PP KO mice compared to virgin KO mice (2.4±0.3 vs. 1.1±0.1 fold change; n=6,5; p=0.0359). GPER1 deletion increased CYP112B expression in virgin mice (2.0±0.4 vs. 1.0±0.2 fold change; n=7,5; p=0.0265), and this was eliminated with PP (0.9±0.2 fold change; n=5; p=0.0260). Aldosterone regulates renal epithelial sodium channel (ENaC), so we assessed its subunits’ expression. Renal ENaCα and ENaCβ increases with the deletion of GPER1 in the virgin group, and this effect was lost with PP. ENaCγ withheld the same trend, but it did not reach significance. Overal, our data suggest that GPER1 and former pregnancy protect from hypertension in aged females at least partially via regulating aldosterone and ENaC signaling.

ODP037 Effects of DHCR24 Inhibitor on Excessive Adrenal Steroidogenesis in db/db mouse

Abstract Background and purpose Patients with type 2 diabetes have an elevated basal level of cortisol, which has been suggested to be involved in the exacerbation of hyperglycemia and the progression of organ complications. Furthermore, it has been reported that adrenal steroidogenesis increases in db/db mice, an animal model of type 2 diabetes. However, the mechanism of the increased adrenal steroidogenesis in type 2 diabetes is unclear. In this study, we aimed to elucidate the mechanism of increased adrenal steroidogenesis in db/db mice and examine whether it is a therapeutic target. Experiment 1: Methods Gene expression in adrenal glands of 10-week-old male db/db mice and db/+ mice was examined by DNA microarray and real-time PCR. Results In the adrenal glands of db/db mice, expression of Acc and Scd1, which are involved in fatty acid synthesis, was decreased by 3.7-fold and 2.6-fold, respectively, compared with db/+ mice. However, expression of Cpt1α, which is involved in acetyl-CoA production by lipolysis, was increased by 1.8-fold and expression of Dhcr24, the final enzyme of the de novo cholesterol synthesis system, was increased by 2.5-fold. Thus, fatty acid degradation and endogenous cholesterol synthesis had increased in the adrenal glands of db/db mice. Experiment 2: Methods 8-week-old male db/db mice were divided into two groups, PBS administration (P) and 10mg/kg DHCR24 inhibitor (U18666A) administration (U) groups. PBS or U18666A was injected intraperitoneally into mice once a week. 4 weeks later, the mice were euthanized, blood corticosterone was measured by LC-MS/MS, and gene expression in adrenal glands was measured by real-time PCR. Results The blood corticosterone level was 196.6±12.7ng/mL in the U group and 245.8±15.4ng/mL in the P group, which was significantly lower in the U group (unpaired t-test, p<0. 05). mRNA expression of Acc and Scd1 was increased by 3.1-fold and 1.3-fold, respectively, in the U group compared with the P group. Conclusion These results suggest that increased endogenous cholesterol synthesis may be a cause of steroid overproduction in the adrenal glands of db/db mice and a DHCR24 inhibitor suppresses the increase in steroid hormone synthesis. This study indicates that changes in adrenal fatty acid metabolism and endogenous cholesterol synthesis might play a major role in understanding the mechanism of increased adrenal steroidogenesis and finding a new therapeutic target to prevent organ complications of type 2 diabetes. Presentation: No date and time listed

PSAT091 The Impact of Pregnancy and Aging on the Female Mouse Adrenal Transcriptome

Abstract Introduction Mouse adrenal glands contain a zona glomerulosa (mineralocorticoid production), zona fasciculata (glucocorticoid production), and a remnant of the fetal adrenal cortex (termed the x-zone). Instead of involuting at birth, as seen in humans, the x-zone expands postnatally and is predicted to function in the metabolism of progesterone and 11-deoxycorticosterone via its high expression of the enzyme, 20α-hydroxysteroid dehydrogenase (Akr1c18). Moreover, mouse adrenals have a clear x-zone sex-divergence at the time of puberty when the x-zone regresses in males. In females, the x-zone regresses during the first pregnancy and with aging. Sexual dimorphism unrelated to the x-zone has been documented in mouse adrenals, including differentially expressed sex-chromosome and sex-steroid driven genes, making the definition of x-zone specific transcripts more challenging. Objective Use RNA sequencing (RNAseq) to better define the x-zone transcriptome by comparing virgin mice to post-pregnant and aged females. Methods Adrenals from four 25-week-old (wk) virgin, four 25 wk post-pregnant, and four 50 wk female mice were used for RNAseq. Reads were aligned to the mm38 assembly with STAR. Expression was quantified with featureCounts. Normalization was performed with edgeR using the TMM method. Unwanted sources of variation were removed with sva. Statistical comparison was performed using limma. Statistically significant (FDR-adjusted, P<0.05) expression between groups allowed characterization of differentially expressed genes (DEGs). Results RNAseq allowed the analysis of adrenal gland RNA transcripts and identified 17,829 genes that met read count thresholds. 246 DEGs were found between virgin and post-pregnant females with 93 showing higher expression in virgin adrenals and 153 with higher expression in post-pregnant female adrenals. The most altered genes included Akr1c18 (fold change (FC) = 174.06; higher in virgin adrenals) and the proton transporter, Otop1 (FC = 20.82; higher in post-pregnant adrenals). When comparing 25 wk to 50 wk adrenals, 269 DEGs were found with 136 showing higher expression in 25 wk adrenals and 133 higher in the 50 wk adrenals. The most altered genes were Akr1c18 (FC = 34.17; higher in 25 wk adrenals) and the mouse mast cell protease, Mcpt2 (FC = 14.42; higher in 50 wk adrenals). The 25 wk virgin mice had 22 transcripts that were higher and 41 that were lower than found in both post-pregnant and 50 wk animals. Within this list are the established x-zone markers: Akr1c18 and the phosphoinositide 3-kinase, Pik3c2g. Discussion Clear differences in transcript levels were observed between virgin mice and those who had underwent pregnancy or aged to 50 wk. Both aged and post-pregnant mice showed lower expression of known x-zone transcripts and RNAseq analysis provided a tentative list of additional x-zone markers. Using female mice with differing x-zone status reduced the number of sexually dimorphic genes and allowed the characterization of genes related to the x-zone. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

ODP018 A Case of Cushing's Syndrome due to Primary Bilateral Macronodular Adrenal Hyperplasia

Abstract Introduction Primary bilateral macro-nodular adrenal hyperplasia (PBMAH) is a rare cause of Cushing's syndrome, characterized by a high clinical heterogeneity, both with regards to the severity of cortisol excess and the morphologic appearance of the adrenals. We present an interesting case of Cushing's syndrome caused by Primary bilateral macro-nodular adrenal hyperplasia. Case presentation A 61-year-old female with Graves’ disease status post I-131 therapy was incidentally found to have two left adrenal adenomas of sizes 2 cm and 1.9 cm on CT imaging. Her laboratory workup revealed Cushing's syndrome evidenced by abnormal 1 mg dexamethasone suppression test (DST) with cortisol of 7.8 mcg/dl (8-19 mcg/dl), 24-hour urinary cortisol (UFC) of 50 mcg/24hr (0-50 mcg/24hr) with ACTH 6.2 pg/ml (7.2 -63 pg/ml) and DHEAS 79.8 mcg/dl (29.4-215 mcg/dl). Her MRI brain revealed meningioma and she underwent gamma knife therapy. Over time she developed uncontrolled hypertension, pre-diabetes, weight gain and easy bruising. On examination she has a plethoric face, ruddy complexion, and central obesity. Her repeat blood work showed 1 mg DST cortisol of 6.7 mcg/dl (8-19 mcg/dl), UFC 76 mcg/dl (6-42 mcg/dl) and normal late night salivary cortisol 0. 071, 0. 067 mcg/dl (<0. 010-0. 090 mcg/dl). Her repeat CT abdomen showed bilateral adrenal nodules with larger nodule on the left side measuring 2.4 cm. She underwent adrenal vein sampling using epinephrine as control which showed bilateral cortisol secretion. She did not tolerate medical therapy with Mifepristone. Testing for aberrant receptors was negative. She eventually underwent left adrenalectomy which improved her clinical co-morbidities and normalized cortisol levels. Her postoperative DST cortisol is 1.5 mcg/dl (8-19 mcg/dl) and UFC is 8 mcg/24hr (6-42 mcg/24hr). Her genetic testing is still pending. Discussion The conventional treatment of PBMAH is adrenalectomy. Unilateral adrenalectomy of larger adrenal gland is now considered a reasonable alternative to bilateral adrenalectomy in selected patients due to risk of adrenal insufficiency and lifetime steroid dependency with bilateral surgery (1). Cortisol secretion in PBMAH can be regulated by aberrant adrenal expression of receptors and all patients should be evaluated for these receptors as there is potential for medical therapy in them. PMBAH is also caused by ARMC5 mutations in familial cases, and it is commonly associated with intracranial meningiomas suggesting a possibility of new syndrome which needs to be further studied. Cranial imaging should be offered to these patients to exclude a coexisting meningioma (2).

OR13-5 Bone Morphogenetic Protein-4 (BMP4) Inhibits Adrenal Cell Cortisol Synthesis by Blocking CYP17A1 and CYP11B1 Expression.

Abstract Background Cytochrome P450c17 (17α-hydroxylase, encoded by CYP17A1) and cytochrome P450c11 (11β-hydroxylase, encoded by CYP11B1) are essential for production of adrenal cortisol. The expression of CYP17A1 and CYP11B1 in adrenocortical cells is stimulated by adrenocorticotropic hormone (ACTH) through the cAMP and protein kinase A signal transduction pathway. Several growth factors can act as autocrine/paracrine regulators for modulating the steroidogenesis in the adrenal cortex. Among these factors, bone morphogenetic protein-4 (BMP4), a member of the superfamily of transforming growth factor-beta (TGFβ) has been previously reported to have an inhibitory effect on adrenal cell CYP17A1 expression and dehydroepiandrosterone (DHEA) production [1]. The role of BMP4 within the adrenal and its mechanisms regulating steroidogenesis are still unclear. In the present study, we examined human adrenal expression of BMP4 and its chronic effects on the H295R adrenal cell line. Objective To define the autocrine/paracrine role of BMP4 in adrenocortical zonation and adrenal steroidogenesis. Methods BMP4 expression in normal human adrenal glands was studied using microarray analysis with laser-captured adrenal capsule and cortical zones. Adrenal BMP4 protein expression was examined following immunohistochemistry (IHC) with a rabbit polyclonal antibody against human BMP4. In vitro studies were performed using H295R and human adrenal cells treated with or without ACTH (10nM), forskolin (10 µM) and BMP4 (50 ng/mL). The experimental medium was collected every 24 h and analyzed for cortisol. Experiments were terminated at 96 h followed by isolation of RNA and qPCR analysis for PPIA (normalization transcript), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), CYP17A1 and CYP11B1. Results Microarray analysis of adrenal zone RNA demonstrated a BMP4 expression gradient with zona glomerulosa (ZG) expressing 3 and 11-fold higher levels than the zona fasciculata (ZF) and zona reticularis (ZR), respectively. IHC confirmed that BMP4 expression was highest in the ZG and the outer ZF. Administration of BMP4 to cultures of H295R cells caused a significant decrease in basal and forskolin-stimulated cortisol production at 48 h. By 96 h, BMP4 inhibited forskolin-stimulated cortisol by 60%. Cortisol inhibition occurred in conjunction with an inhibition in forskolin-stimulated CYP17A1 (↓75%) and CYP11B1 (↓65%). However, CYP11A1 expression was unaffected. In primary cultures of human adrenal cells BMP4 inhibited the ability of ACTH to increase cortisol production (↓75%), as well as CYP11B1(↓65%) and CYP17A1 (↓57%) expression. Conclusion Our findings indicate that the human adrenal has outer cortex localized expression of BMP4 that could play a paracrine/autocrine role in zone-specific production of aldosterone vs. cortisol through BMP4 selective inhibition of CYP17A1 and CYP11B1. Bibliography: 1. Rege J, Nishimoto HK, Nishimoto K, Rodgers RJ, Auchus RJ, Rainey WE. Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis. Endocrinology 2015; 156: 2530–2540. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

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.

Endogenous Cushing's syndrome during pregnancy.

Endogenous Cushing's syndrome (CS) is rare during pregnancy, probably because hypercortisolism induces anovulation and infertility. To date, slightly above 200 cases have been reported in the literature. The most frequent etiology of CS diagnosed during gestation is from primary adrenal causes, namely adrenal adenomas and an entity called pregnancy-induced CS. The latter can be secondary to the aberrant adrenal expression of luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) in the adrenal lesions. Diagnosis of CS during pregnancy is extremely challenging, as a consequence of the physiologic hypercortisolism normally present during pregnancy. Assessment of excess cortisol production tests should be interpreted cautiously using adapted upper limits of normal criteria for pregnant patients and a high index of suspicion is required for diagnosis. Imaging is also limited due to high risk of radiation exposure with computed tomography and teratogenicity with contrast agents. The optimal treatment strategy is surgical resection of adrenal adenoma or pituitary adenoma, ideally before 24 weeks of gestation to reduce the risk of maternal and fetal complications. In mild cases, surgery can be postponed until after delivery and treatment should focus on controlling metabolic complications of hypercortisolism, such as hypertension and dysglycemia. Maternal and fetal outcomes of excess cortisol exposure, except fetal loss, are not readily improved by successful treatment of hypercortisolism.

CHANGES OF THE ZONA GLOMERULOSA IN HUMAN ADRENALS FROM BIRTH TO 40 YEARS OF AGE

Objective: Antibodies to CYP11B2 (aldosterone synthase) allow imaging of the zona glomerulosa (zG) by immunohistochemistry. Studies demonstrate that the area and continuity of the zG decreases with aging and accumulations of aldosterone-producing cell clusters occur. Research on age-related changes in the zG in younger patients and comparison of both adrenals is limited. Therefore, the objective of this study was to elucidate age-related changes of the zG by quantifying CYP11B2 expression in adrenals of patients below the age of 40 years. Design and method: We collected human adrenals from autopsy cases, that were anonymized except for age, sex and medical history. We excluded adrenal glands of patients with premature birth, hypertension, cardiovascular disease, adrenal tumors, use of steroids. In case of two available adrenals in one patient, one was randomly selected of the two. We validated software for automated quantification by comparing manually outlined CYP11B2 expressing area with automated outlined area. We then used this automated method to quantify CYP11B2 expressing area, CYP11B2 continuity and mean and maximum CYP11B2 depth. If the zG was discontinuous, mean length of ‘gaps’ between ZG tissue was calculated. We also determined the structural similarity between both adrenals of the same case. The study was approved by the local Medical Ethics Committee. Results: There was a negative correlation between age and CYP11B2 expressing area (rs = -.63); negative correlation between age and CYP11B2 continuity (rs = -.80); positive correlation with age and mean gap length (rs = 0.71); positive correlation between age and maximum CYP11B2 depth (rs = .30, P = <0.001) and no correlation between age and mean CYP11B2 depth (rs = .01, p = <0.001). The intraclass correlation coefficient between both adrenals of one subject for CYP11B2 expressing area was 0.78 (95%CI = 0.56–0.89), for CYP11B2 continuity 0.94 (95%CI = 0.88–0.97) and for mean gap length 0.76 (95%CI = 0.32–0.81). Conclusions: We demonstrate a decrease in CYP11B2 area and continuity, whilst the mean gap length and maximum CYP11B2 depth increases, which we consider as involution and clustering of the zG. This is seen in both adrenals and therefore suggests a common process. The relation with the development of aldosterone-producing adenoma remains to be determined.

In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3β-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

Adrenal gland responses surgical castration and immunocastration by different compensatory manners to increase DHEA secretion

Androgen from the testis and weak androgens from the adrenal cortex may interact with each other and affect their synthesis and secretion due to their similar functions. The purpose of this study was to investigate the compensatory effect of adrenal in rats after immunocastration and surgical castration, and the interaction between the hypothalamic–pituitary–testis (HPT) axis and the hypothalamic–pituitary–adrenal (HPA) axis. 24 male SD rats aged 8 weeks were randomly divided into three groups and accepted treatments: surgical castration group, immunocastration group and control group. In both surgical castration and immunocastration groups, the secretion of adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone (DHEA) hormones was significantly increased compared with the control group (p < 0.05). In the HPT axis of the immunocastration group, the KISS1 expression was up-regulated, whereas GPR54, LH and LHR expression were down-regulated (p < 0.05). The expression levels of CRH, POMC and MC2R genes were also significantly up-regulated (p < 0.05). In addition, in the immunocastration group, the expression of adrenal LHR mRNA expression was decreased (p < 0.05). The expression of HPT axis genes and adrenal LHR were up-regulated in the surgical castration group (p < 0.05). These results show that in both immunocastration and surgical castration, adrenal androgen is increased, suggesting that the adrenal gland plays a compensatory role. Moreover, it also shows that different castration treatments have effects on adrenal steroid secretion through different mechanisms.

An Ethanolic Extract of Cucurbita pepo L. Seeds Modifies Neuroendocrine Disruption in Chronic Stressed Rats and Adrenal Expression of Inflammatory Markers and HSP70.

Background: Pumpkins (Cucurbita pepo L.) were described to have antioxidant, anti-inflammatory, anti-fatigue, and antidepressant-like effect. The adrenal gland is an important stress-responsive organ that maintains homeostasis during stress. Objectives: This study aimed to assess the efficacy of the administration of Cucurbita pepo L. (CP) extract in relieving behavioral, biochemical, and structural changes in the adrenal gland induced by exposure to chronic unpredictable mild stress (CUMS) and to explore the mechanism behind this impact. Materials and Methods: Forty male albino rats were divided into 4 groups (n = 10): control, CUMS, fluoxetine-treated, and CP-treated groups. Behavioral changes, corticosterone level, pro-inflammatory cytokines TNF-α and IL-6, and oxidant/antioxidant profile were assessed in the serum at the end of the experiment. Adrenal glands were processed for histopathological and immunohistochemical assessment. Gene expression of caspase-3 and Ki67 and heat shock protein 70 (HSP70) were assessed in adrenal glands using RT-PCR. Results: The CP extract significantly reduced the corticosterone level (p < 0.001), immobility time (p < 0.001), and inflammatory and oxidative changes associated with CUMS-induced depression compared to the untreated group. The CP extract alleviated CUMS-induced adrenal histopathological changes and significantly reduced apoptosis (p < 0.001) and significantly upregulated antioxidant levels in the serum. Conclusion: Cucurbita pepo L. effectively ameliorated the chronic stress-induced behavioral, biochemical, and adrenal structural changes mostly through its antioxidant and anti-inflammatory effects.

Abstract 10: MANP Activation of the Cyclic GMP/PDE2/CYP11B2 Pathway Inhibits Aldosterone In Vitro and In Vivo

Aldosterone is a critical driver for cardiovascular disease (CVD). We recently discovered that MANP, a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP. MANP is currently entering clinical trials for hypertension and thus understanding its aldosterone suppressing mechanism is important. The mechanism of aldosterone inhibition by natriuretic peptides (NPs) remains to be clearly defined. Conflicting results were reported on the roles of particulate guanylyl cyclase A receptor (pGC-A) and NP clearance receptor (NPRC) in aldosterone inhibition. Furthermore, the functions of protein kinase G (PKG) and phosphodiesterases (PDE) on aldosterone regulation are not clear. Herein, we investigated the molecular mechanism of aldosterone regulation in the human adrenocortical cell line H295R and in mice. We firstly showed that pGC-A mediates aldosterone inhibition. In contrast, with NPRC agonist and antagonist, we showed that NPRC did not inhibit aldosterone. Next, we confirmed that MANP inhibits aldosterone via PDE2, not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer (FRET) experiments. Specifically, MANP suppressed ANGII mediated activation of aldosterone (fold change) MANP+ANGII 3.2±0.1* vs. ANGII 3.8±0.1 (*p&lt;0.05) with IBMX, a PDEs inhibitor and the PDE2 antagonist Bay 60-7550 reversed MANP-mediated aldosterone suppression (IBMX+MANP+ANGII 3.9±0.2 and Bay+MANP+ANGII 4.1±0.1). With PKG agonists and inhibitors, aldosterone levels were not changed. In PDE2 activity FRET studies, aldosterone control was 3.7±0.4 and with MANP 0.9±0.2* supporting PDE2 activation by MANP. Further, the inhibitory effect of PDE2 is mediated by a reduction of intracellular Ca2+ concentration (~22%). We then showed that MANP directly reduced aldosterone synthase CYP11B2 expression in vitro. Lastly, in PDE2 knockout mice (embryonic lethal), embryonic adrenal CYP11B2 expression is markedly increased (wild type: 1±0.2, KO: 2.8±0.5*). Our findings innovatively elucidate the pGC-A/cGMP/PDE2 pathway in aldosterone inhibition by MANP in vitro and in vivo. Additionally, our data also support the development of MANP as a novel ANP analog drug for CVD.

Approaches to Gene Mutation Analysis Using Formalin-Fixed Paraffin-Embedded Adrenal Tumor Tissue From Patients With Primary Aldosteronism.

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal’s capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.

UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.