Zona Fasciculata Research Articles

OR08-03 Ectopic Localization Of CYP11B1 and CYP11B2-Expressing Cells In The Normal Human Adrenal Gland

Abstract Disclosure: C. Duparc: None. P. Camponova: None. M. Roy: None. H. Lefebvre: None. M. Thomas: None. The zonation of the human adrenal cortex has long been established morphologically and histologically as three distinct layers of cells. The outer zona glomerulosa (ZG) comprises densely packed cells arranged in clusters that produce aldosterone; the zona fasciculata (ZF) is composed of cells with large cytoplasm, containing lipid droplets arranged in radial columns that synthetize cortisol; and the zona reticularis (ZR) is composed of compact and pigmented cells producing androgens. The sharp line of demarcation between ZG and ZF has been recently challenged suggesting that this interface is no longer a compartment boundary. We have used immunohistochemical analyses to study the steroid 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) pattern of expression and investigate the remodeling of the adrenal cortex in relation to aging. We analyzed human adrenal glands prepared from 47 kidney donors (18 females, 29 males, ranging in age from 22 to 81 years old). No aldosterone-producing micronodules (APMs) were detectable in the younger donors aged between 22-39 but the functional ZG depicted by positive CYP11B2 staining demonstrated a lack of continuity. In contrast, the development of APMs was found in samples from individuals aged 40-70. None of the 47 adrenals studied had a continuous CYP11B2 expression which appeared to be fragmented within the subcapsular region. Importantly, the progressive replacement of CYP11B2-expressing cells in the histological ZG by CYP11B1-expressing cells highlights the remodeling capacity of the adrenal cortex. Cells expressing respectively CYP11B1 and CYP11B2 were found interwoven at different levels: i) single or cluster of CYP11B2 cells can be observed in the inner cortex surrounded by CYP11B1-expressing cells (33/47, 70.21%) and ii) the two type of cells might be found in the same nest or basket of cells beneath the capsule (40/47 donors, 85.10%). Interestingly, CYP11B1 and CYP11B2 cells were also present within the ZR. Proliferation detected by Ki-67 expression occurred in 14.04% (±1.69%) of the CYP11B2-expressing cells while it appeared expressed in 85.96% (±1.69%) of the CYP11B1-positive cells (p<0.001). No significant correlation between Ki-67 and age or sex has been found. Our data emphasize that mineralocorticoid- and glucocorticoid-producing cells are distributed throughout the cortex and the medulla making the determination of the functional status of a cell or group of cells a unique tool in deciphering the changes occurring in adrenal gland particularly during aging. Despite the fact that our data cannot exclude the existence of the CYP11B2+ cell conversion/migration lineage as demonstrated in mice they suggest that alternative mechanisms may exist in the human adrenal such as inward displacement of CYP11B2 expressing cells without lineage conversion. Presentation: Friday, June 16, 2023

OR27-05 Two-pronged Mediation Of Adrenal Steroidogenesis By Oxytocin

Abstract Disclosure: U. Cheliadinova: None. G. Pevnev: None. V. Ryu: None. T. Frolinger: None. S.L. Sims: None. M. Ofer: None. F. Korkmaz: None. O. Barak: None. J. Gimenez Roig: None. F. Sultana: None. N. Kramskiy: None. S. Wizman: None. M. Orloff: None. T. Yuen: None. D. Lizneva: None. M. Zaidi: None. A. Gumerova: None. Stress–stimulated glucocorticoid release affects diverse biological processes, including pregnancy and childbirth. The controversy surrounding effects of OXT on stress prompted us to investigate its actions on adrenal cortical function in mice. RNAscope and immunohistochemistry provided unequivocal evidence for abundant OXT receptor (OXTR) expression in the adrenal cortex, predominantly in zona fasciculata and zona reticularis. Female mice displayed higher adrenal cortical Oxtr expression, more than a range of other tissues under study. Oxtr expression was also detected in the adenohypophysis solely in female mice, while both male and female mice expressed Oxtr in the pars intermedia of the hypophysis. Aging impressively attenuated Oxtr expression in female mice in both the adrenal gland and adenohypophysis. For loss–of–function studies, we generated tissue–specific Star-CreERT2/Oxtrfl/fl mice in which the Oxtr was deleted from steroidogenic tissues, namely adrenals and ovaries. Deletion was confirmed by RNAscope and immunohistochemistry. We found no difference in serum corticosterone between tamoxifen–induced and uninduced mice; however, the elevation of serum corticosterone upon ACTH stimulation or after four weeks of stress was attenuated in tamoxifen–induced mice. Bulk RNAseq of adrenals from tamoxifen–induced mice revealed the downregulation of key steroidogenic genes, namely Cyp17a1, Hsd3b3, Hsd17b2, Cyp3a41b, and Serpina6 (a corticosteroid–binding globulin). In gain–of–function studies, a single OXT injection led to a rise of serum ACTH in tamoxifen–induced mice, which was greater in magnitude than uninduced controls. However, after five days of OXT injections, corticosterone levels became elevated, whereas ACTH levels declined to baseline. This suggests that OXT acts directly on OXTRs on corticotropes to initiate a stress–like response, with a negative feedback loop that inhibits further ACTH secretion. Of note is that male mice did not display any difference between tamoxifen–induced and uninduced groups, consistent with their low hypophyseal Oxtr expression. In all, we describe a novel gender–specific two–pronged circuit through which OXT may precisely regulates the production of corticosterone from the adrenal cortex. In the first arm, OXT directly stimulates ACTH secretion through adenohypophysis OXTRs. In the second arm, OXT prevents excessive stress–induced adrenal stimulation by downregulating steroidogenic genes. Our discovery lays the foundation for the emergence of new physiology that may relate to a fundamental role for oxytocin in the stress response during procreation, notably, when levels are elevated during pregnancy and lactation. Presentation: Saturday, June 17, 2023

OR08-01 Expression of Gonadal Differentiation Markers in Bilateral Macronodular Adrenocortical Disease

Abstract Disclosure: K. De Sousa: None. V. Pautasso: None. C. Duparc: None. S. Renouf: None. B. Ragazzon: None. F. Violon: None. L. Bouys: None. F. Bonnet-Serrano: None. M. Sibony: None. N. Driessens: None. S. Espiard: None. G. Raverot: None. J.Y. Bertherat: None. E. Louiset: None. H. Lefebvre: None. Context: Bilateral macronodular adrenocortical disease (BMAD) is a rare cause of primary Cushing syndrome, occurring mostly in women. ARMC5 and KDM1A mutations are responsible for distinct forms of BMAD. In particular, KDM1A mutations are associated with food-dependent Cushing syndrome. In addition, BMAD tissues can express illegitimate receptors, including LH receptor, that lead to aberrant control of cortisol secretion by various factors. Moreover, steroidogenic cells are able to abnormally produce bioactive factors, such as ACTH and the Leydig cell hormone INSL3. Since LH receptor, ACTH and INSL3 are known to be produced in gonads, we speculated on a gonadal-like differentiation of adrenocortical cells in BMAD tissues. Aim: The aim of our study was to investigate the presence of different gonadal markers in a series of BMAD tissues, and to search for secretion of adrenal and gonadal hormones by BMAD cells. Methodology: Gene expression of gonadal markers were evaluated by RNAseq. Immunohistochemical studies were performed on 41 BMAD and 9 normal adrenal samples to examine the presence of hormones (ACTH, INSL3, anti-Mullerian hormone [AMH]), the estrogen synthetizing enzyme aromatase, and transcription factors (GATA4, SOX9, FOXL2, MAGEA4) known to be expressed in testis or ovary. Perifusion experiments, coupled to immunoassays or liquid chromatography/mass spectrometry, were conducted to quantify hormone secretion by 2 adrenal explants. Results: BMAD tissues were collected from patients (60% women) without or with ARMC5 (32%) or KDM1A (13%) mutations. ARMC5-mutated tissues overexpressed FOXL2 and CYP19A1 (encoding aromatase) in comparison with other BMAD samples. In normal adrenals, cortisol-producing cells, located in the zona fasciculata, were immunonegative for INSL3, AMH, aromatase, GATA4 and SOX9, whereas FOXL2 and MAGEA4 immunostainings were detected in the cortex. In BMAH tissues, the presence of positive cells for ACTH (100% of BMAD), INSL3 (90%), AMH (50%), aromatase (77%), SOX9 (92%), GATA4 (97%), FOXL2 (100%) and MAGEA4 (100%) were observed in most of the samples regardless of the gender or mutational status. FOXL2 and MAGEA4 immunostainings were detected throughout BMAD tissues. Distribution of other gonadal markers was heterogenous in tissue sections with clusters of cells positive for ACTH, aromatase and GATA4 or ACTH and SOX9. Functional studies revealed pulsatile secretion of different glucocorticoids and INSL3 by BMAD explants. ACTH and hCG stimulated steroid production but were without effect on INSL3 release. Conclusion: These data show that steroidogenic cells in BMAD tissues exhibit a gonadal-type phenotype and abnormally secrete INSL3. They suggest that BMAD may result from a genetically driven defect occurring during early embryogenesis and affecting differentiation and development of adrenal tissues. Presentation: Friday, June 16, 2023

FRI139 Identification Of Robust Aldosterone-Producing Adenomas-related Genes Using Adrenal Genome-wide Transcriptome Analysis

Abstract Disclosure: S. Lee: None. T. Lee: None. G. Yoo: None. J. Lim: None. Primary aldosteronism (PA), the most common cause of secondary hypertension, is caused by aldosterone overproduction in one or two adrenal glands. It carries a higher risk of cardiovascular disease and mortality compared to essential hypertension. In an effort to identify the underlying molecular mechanisms regarding aldosterone secretion and cell proliferation in aldosterone-producing adenomas (APA), genome-wide transcriptome analyses have been conducted; however, there are still some limitations that need to be addressed since a relatively small number of samples were analyzed at a single center in most studies. In this study, we performed integrative and systematic analysis using adrenal transcriptome datasets to verify candidate genes robustly associated with APA and obtain more generalized signatures involved in the pathophysiology of PA. Three mRNA datasets for adrenal tissues (GSE8514, GSE60042, and GSE64957) were obtained from the Gene Expression Omnibus Database. For the first two datasets, normal adrenal samples from patients with renal cell carcinoma and adrenal tissue samples adjacent to APA were included as controls, respectively. In GSE64957, zona glomerulosa (ZG) and zona fasciculata (ZF) samples were analyzed separately in the current study (GSE64957ZG and GSE64957ZF). Differentially expressed gene (DEG) investigation and comparative analysis were conducted using the limma method. The selection criteria for APA-related genes were as follows: those that are significantly dysregulated in more than three of the four datasets and show a consistent trend of up- or down-regulation in four datasets. 5,375 genes were present in all transcriptome datasets; for the GSE64957ZG, 57 DEGs were identified in the APA group, among which 14, 7, and 37 genes were also dysregulated in the APA group of GSE64957ZF, GSE8514, and GSE60042, respectively. All comparisons among four lists of DEGs exhibited significant enrichment results. The highest degree of overlap was shown in the DEG pair of GSE64957ZG-GSE60042. As a result, approximately 20 genes (12 upregulated and 12 downregulated DEGs), including CYP11B2, PCP4, and ATP2A3, were selected as the final APA-related genes. This study demonstrated several candidate genes that are strongly relevant to APA by examining whole genome disease-related signatures. Besides CYP11B2, it seems that alteration in PCP4 and ATP2A3 genes would be much more involved in the pathogenesis of PA, which warrants further investigation along with validation for the role of the remaining genes. Presentation: Friday, June 16, 2023

FRI166 Characterization of Aldosterone-producing Cell Cluster (APCC) At Single-cell Resolution

Abstract Disclosure: N. Iwahashi: None. H. Umakoshi: None. T. Seki: None. C. Gomez-Sanchez: None. K. Mukai: None. M. Suematsu: None. Y. Umezawa: None. M. Oya: None. T. Kosaka: None. M. Seki: None. Y. Suzuki: None. Y. Horiuchi: None. K. Nishimoto: None. Y. Ogawa: None. Context: The adrenal cortex consists of zona glomerulosa (ZG), fasciculata (ZF), and reticularis. Aldosterone-producing cell clusters (APCCs) that express aldosterone synthase (CYP11B2) strongly can be frequently found in adult adrenals and harbor somatic mutations that are also present in aldosterone-producing adenomas (APAs). Primary aldosteronism is mainly caused by APAs or idiopathic hyperaldosteronism (IHA). We presume that APCCs are causing IHA and are precursors of APAs. However, the gene expression characteristics and especially the development of APCCs are not well understood. Objective: This study aimed to analyze the transcriptome of APCCs at single-cell resolution and infer the developmental trajectory. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on two adult adrenals. Results: Immunohistochemical analyses confirmed the presence of APCCs in the two adrenals. scRNA-seq data of 6770 adrenal cells were obtained and 2928 high-quality cells were selected after quality control procedures. Unsupervised clustering and marker gene expressions such as CYP11A1 and NR5A1 identified 1765 adrenocortical cells, which were further divided into six clusters (B1-6). Using the list of APCC/ZG upregulated genes from our previously reported DNA microarray study, three clusters (B1, B3, and B6, totaling 923 cells) were identified as APCC/ZG cells. By further subclustering, the APCC/ZG cells were divided into 3 clusters (C1, C2, and C3). The APCC cluster (C3, 64 cells) and ZG cluster (C1, 447 cells) were identified. Cluster C2 seemed to be ZG-to-ZF transitional cells. RNA velocity analysis, which aimed to infer the direction of differentiation, inferred a direction of development from ZG cells to APCCs. Conclusion: Our results revealed the gene expression characteristics of APCC at single-cell resolution and show that some ZG cells remodel into APCCs. Presentation: Friday, June 16, 2023

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

Effect of Bee Bread on Corticosterone Level in Rat Dams Exposed to Gestational Heat Stress.

Environmental temperature rises are powerful stimuli that can alter both the sympathetic nervous system and the hypothalamic-pituitary-adrenocortical axis (HPA). Heat stress has been shown to harm pregnancy outcomes such as causing spontaneous abortion, low birth weight, growth retardation and stillbirth. Supplementation of bee bread in pregnant rats under heat stress exposure has been shown to improve the pregnancy outcomes. However, whether supplementation of bee bread during heat stress exposure may also reduce the level of the stress hormone, corticosterone has yet been reported. Therefore, this study aims to determine the effect of bee bread on corticosterone level, progesterone level, oestradiol level and zonation of the adrenal cortex of pregnant rats under heat stress exposure. Pregnant rats were randomly categorised into four groups (n = 6): Control (C: standard feeding), Treatment 1 (T1: 0.5 g bee bread/kg body weight/day), Treatment 2 (T2: standard feeding with heat exposure), and Treatment 3 (T3: 0.5 g bee bread/kg body weight/day with heat exposure). Bee bread (0.5 g/kg body weight/day) was force-fed to pregnant rats through oral gavage beginning on day 0 of pregnancy and continuing until delivery. Heat stress was generated experimentally by putting both T2 and T3 rats in an egg incubator for 45 min daily at a temperature of 43°C till delivery. On a postnatal Day 21, dams were euthanised to assess serum corticosterone, progesterone, oestradiol levels and adrenal gland histology. Rats in the T2 group had a significantly (P < 0.05) increase in the zona fasciculata thickness (94.95 ± 1.55 μm) and higher corticosterone levels (49.57 ± 1.57 ng/mL) compared with control. However, supplementation of bee bread during heat stress was able to show an improvement in adrenal zona fasciculata thickness by decreasing to 79.89 ± 3.08 μm and corticosterone level reduced to 35.31 ± 1.73 ng/mL significantly (P < 0.05). Therefore, these findings may imply that bee bread is effective as a neutralizer in lowering the production of stress hormone.

Transplantation of porcine adrenal spheroids for the treatment of adrenal insufficiency.

Primary adrenal insufficiency is a life-threatening disorder, which requires lifelong hormone replacement therapy. Transplantation of xenogeneic adrenal cells is a potential alternative approach for the treatment of adrenal insufficiency. For a successful outcome of this replacement therapy, transplanted cells should provide adequate hormone secretion and respond to adrenal physiological stimuli. Here, we describe the generation and characterization of primary porcine adrenal spheroids capable of replacing the function of adrenal glands in vivo. Cells within the spheroids morphologically resembled adult adrenocortical cells and synthesized and secreted adrenal steroid hormones in a regulated manner. Moreover, the embedding of the spheroids in alginate led to the formation of cellular elongations of steroidogenic cells migrating centripetally towards the inner part of the slab, similar to zona Fasciculata cells in the intact organ. Finally, transplantation of adrenal spheroids in adrenalectomized SCID mice reversed the adrenal insufficiency phenotype, which significantly improved animals' survival. Overall, such adrenal models could be employed for disease modeling and drug testing, and represent the first step toward potential clinical trials in the future.

Age Related Histoarchitectural Studies on the Adrenal Gland of Goat (Capra hircus)

Background: Adrenal gland is an important endocrine gland that secrets several hormones that play a key role in the adaptation of the newborn to extra uterine life. The study was conducted to explore the histomorphological development of adrenal glands in Gohilwadi Goat (Capra hircus). Methods: The research work was carried out on twenty pair of goat adrenal gland collected from local abattoir. The animals were divided into three age groups, viz. group- I (day old to 1 month), group- II ( greater than 1 month to 6 months) and group- III ( greater than 6 months). Result: Histological observations revealed that the gland was fully differentiated into cortex and medulla, completely surrounded by a dense fibrous capsule in the day old goats. In group- I, adrenal cortex was made up of definite and foetal cortex, whereas former one comprised of zona glomerulosa and zona fasciculata only. Small to elongate foetal cortical cells were characterized by eosinophilic cytoplasm and arranged into irregular cords. In group II, the foetal cortex disappeared and only definite cortex was present with well-developed three zones i.e. Zona glomerulosa, zona fasciculata and Zona reticularis. Zona glomerulosa was the smallest of the three cortical zones and thickness increased from group-I to group-III. The zona fasciculata was the largest among the three cortical zones in all the age groups and thickness increased from group-I to group-III. Zona reticularis was well organized in the adrenal gland of adult Gohilwadi goat. Medulla occupied greater proportion of the gland and inner medullary cells were the main cellular constituent of medulla. Thickness of medulla increased from group-I to group-III.

New insights into signal transduction pathways in adrenal steroidogenesis: role of mitochondrial fusion, lipid mediators, and MAPK phosphatases.

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

Morphological Harbingers of ARMC5-Pathogenic Variant-Related Bilateral Macronodular Adrenocortical Disease.

Bilateral macronodular adrenocortical disease (BMAD) is a neoplastic disease associated with a high frequency of germline disease. Armadillo repeat containing 5 (ARMC5) pathogenic variants (PVs) have not been widely studied to determine the morphological and immunohistochemical characteristics of BMAD. We carried out a detailed morphologic review of 22 surgical specimens excised from patients with BMAD and compared them with PV of ARMC5 (PV + , n = 14) and those without (PV - , n = 8), and further comparing them with a control group of adrenals excised from patients with renal cancer (n = 11). No patients presented with a genetic syndrome related to BMAD. Overt Cushing's syndrome was present in 12/22 patients, 10 PV + and 2 PV - (p = 0.074). We also evaluated the expression of Ki-67, BCL-2, BAX, p53, CYP11B1, and ARMC5 protein. The pseudo-glandular and trabecular architectural patterns were strongly associated with the PV + group (both p < 0.001), as well as capsular extrusion (p < 0.001). There was no predictive value in the distinction of ARMC5 variants in Hsiao subtyping. ARMC5 diffuse cytoplasmic staining was observed in all 11 control samples. The ARMC5 expression was significantly lower in BMAD than in the control group (p < 0.001). In all the specimens, expression of BCL-2 was identified only in the medulla, and expression of BAX was observed in adrenocortical cells. CYP11B1 diffuse immunoexpression was identified in all the specimens of BMAD and in the fasciculata zone in the control group. The mitotic count and Ki-67 proliferation index was very low in all three groups (controls, PV + , and PV - BMAD). None of the specimens stained positive for the p53 protein. Although our series is limited, the presence of pseudo-glandular and/or trabecular patterns and capsular extrusion indicated the presence of pathogenic variants of ARMC5 in BMAD. The gland enlargement does not seem to be related to the increase of mitotic count or a higher proliferation index (Ki-67).

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.

S-55-4: HISTOPATHOLOGY-GENOTYPE CORRELATION OF ALDOSTERONE- PRODUCING ADENOMAS

Primary aldosteronism (PA) is a vastly underdiagnosed (&lt;1 in 500) cause of secondary hypertension in which aldosterone production is inappropriately high relative to suppressed plasma renin levels. This is of major concern as untreated patients are at increased risk for cardiovascular diseases, stroke, and other comorbidities even when compared to matched essential hypertension patients. PA is commonly caused sporadically by aldosterone producing adenomas (APAs) that harbors somatic mutations in aldosterone driver genes. It can account for up to 5 to 10% of hypertensive cases or up to 10 to 20% of resistant hypertensive cases. The current recommended treatment for APAs when only affecting one adrenal is a unilateral adrenalectomy. This has allowed the excised tissue to be extensively studied and characterized. Interestingly APAs have been found to occur with a spectrum of cell morphology and clinical phenotype. This heterogeneity seem to have a genetic basis. KCNJ5 mutant APAs, the most common genotype of APAs, have zona fasciculata (ZF) like cell morphology &amp; secrete hybrid steroids. They commonly are large and frequently occur among young females and Oriental patients. Whereas CACNA1D mutant APAs are smaller, more zona glomerulosa (ZG) like (i.e. more compact than ZF), and more common in Black patients. To note, CACNA1D is also the most common aldosterone driver mutations in aldosterone producing micronodules and elderly patients, followed by aldosterone driver mutations in ATP1A1 and ATP2B3. CTNNB1 mutant APAs that also harbor GNA11/GNAQ mutations have cells that seem to dedifferentiate towards the adrenal-gonadal precursor cell type as they express the LH/HCG receptor ten fold more than other mutant APAs or even CTNNB1 mutant only APAs. This would explain why patients with the double mutations often present at times of high LH/HCG. These distinct genotype specific phenotypes could be used to facilitate screening and diagnosis of PA. For example, hybrid steroid measurements could be used for screening of KCNJ5 mutant APAs; or onset of hypertension in the first trimester (the period of peak HCG secretion), compared to pregnancy associated hypertension in later trimesters, should prompt consideration for a double mutant APA. Moreover, identification of these somatic mutations has already led to potential therapeutic treatments. For example, chemical screens have identified macrolides that selectively inhibit mutant KCNJ5. Thus, histopathology genotype correlation of APAs have provided new opportunities for the screening, diagnosis, management, and treatment of PA. Further elucidations of the cellular mechanisms associated with each correlation will help to identify the pathological and physiological pathways, and thus novel therapeutic targets.

The human glucocorticoid receptor.

Glucocorticoids are members of steroid hormones that are biosynthesized in the intermediate cellular zone of the adrenal cortex (zona fasciculata) and released into the peripheral blood as final products of the hypothalamic-pituitary-adrenal (HPA) axis, as well as under the control of the circadian biologic system. These molecules regulate every physiologic function of the organism as they bind to an almost ubiquitous hormone-activated transcription factor, the glucocorticoid receptor (GR), which influences the rate of transcription of a huge number of target genes amounting to up to 20% of the mammalian genome. The evolving progress of cellular, molecular and computational-structural biology and the implication of epigenetics in every-day clinical practice have enabled us a deeper and ever-increasing understanding of how target tissues respond to natural and synthetic glucocorticoids. In this chapter, we summarize the current knowledge on the structure, expression, function and signaling of the human glucocorticoid receptor in normal and pathologic conditions.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

S-13-5: UPDATES ON FAMILIAL HYPERALDSOTERONISM

The first reported (Sutherland et al. 1966) form of familial primary aldosteronism (PA) involved a father and son and was glucocorticoid-remediable (familial hyperaldosteronism type I, FH-I). In 1992, Lifton and colleagues elucidated its causation, a hybrid gene mutation composed of CYP11B1 (encoding 11beta-hydroxylase) regulatory sequences and CYP11B2 (aldosterone synthase) coding sequences causing ACTH-regulated aldosterone overproduction and hypertension, both readily controlled by low-dose glucocorticoid administration. Expression of aldosterone synthase activity within zona fasciculata (ZF) explained overproduction of hybrid steroids, 18-hydroxy- and 18-oxocortisol. Since 2011, PA-causing germline mutations in another four genes (three associated with FH) have been reported. While three encode cation channels (KCNJ5, CACNA1H and CACNA1D), one encodes an anion channel (CLCN2). FH-III is caused by inheritance of gain-of-function mutations in KCNJ5, encoding an inwardly rectifying potassium channel (GIRK-4). Depending on the exact mutation involved, phenotype varies from very severe (profound PA, hybrid steroid elevation and ZF hyperplasia and requiring bilateral adrenalectomy to control hypertension) to mild (absence of above with PA controllable with spironolactone). Most pedigrees demonstrate early-onset hypertension. Mutations reduce selectivity of GIRK-4 to potassium, permitting entry of sodium into the adrenal cortical cell, which results in cell depolarization and calcium influx, leading to increased expression of CYP11B2 and aldosterone synthesis. A gain-of-function mutation in CACNA1H, encoding a T-type voltage-gated calcium channel, was found in five of 40 subjects with early-onset PA and three relatives with early-onset hypertension (FH-IV). Adrenals appeared normal on imaging. The mutation shifts activation of zona glomerulosa (ZG) cells to a more hyperpolarized potential and slows inactivation, putatively resulting in increased calcium influx and aldosterone production. Germline mutations in CACNA1D, encoding an L-type voltage-gated calcium channel highly expressed in ZG, were identified in two of 100 individuals with early-onset PA. Both occurred de novo, and were associated with seizures and a variety of neurological abnormalities (PASNA). Both mutations cause channel activation at less depolarized membrane potentials, and one nearly abolished channel inactivation. In a large Australian family with FH-II, a mutation was found in CLCN2 encoding a chloride channel, ClC-2. CLCN2 mutations were found in another seven of 80 individuals with early-onset PA. A concurrent report found a CLCN2 mutation in one of 12 subjects with early-onset PA. PA due to CLCN2 mutations responds well to medical treatment. In the adrenal, ClC-2 localizes predominantly to ZG and mutations predispose cell depolarization, leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production.

Evaluation of Serum Cortisol Levels in Patients with Hypothyroidism at a Tertiary Care Hospital, Telangana, India: A Case-control Study

Introduction: Serum Cortisol is a glucocorticoid hormone released from the zona fasciculata layer of the adrenal cortex, which plays an important role in regulation of blood pressure and metabolism. Hypothyroidism causes elevated cortisol levels due to both decreased clearance and negative feedback of cortisol on the hypothalamic pituitary-adrenal axis. Aim: To investigate the correlation of serum cortisol and with Triiodothyronine (T3), Thyroxine (T4), Thyroid Stimulating Hormone (TSH) and serum cortisol levels in patients with hypothyroidism. Materials and Methods: Present case-control study was carried out in the Department of Biochemistry, Gandhi Medical College and Hospital, Secunderabad, Telanagana, India, July 2022 to August 2022. A total of 60 patients with hypothyroidism (cases) aged between 35-60 years, and 60 controls were selected from Gandhi Medical College and Hospital. Samples were analysed in Siemens Advia centaur XPT. T3, T4, TSH and cortisol levels were estimated by “CLIA” method. Data were statistically analysed using Karl Pearson’s correlation test. Results: In present study, there were five males and 55 females in control group, 23 males and 37 females were in case group. The mean age in cases was 39.39±6.44 years and in control group was 39.38±10.97 years. The mean T3 in subjects cases was 10.74±5.34 ng/mL, as compared to 8.93±2.78 ng/mL, in controls. The mean TSH level in cases was 13.38±20.43 mIU/L, as compared to 3.47±4.20 mIU/L, in controls. The mean cortisol level in cases was 64.88.44±16.25 µg/dL, as compared to 9.55±5.59 µg/dL, in controls. There was significant positive correlation between serum cortisol level and T3 (r=0.12, p-value 0.02), T4 (r=0.12, p-value 0.02), and TSH Levels ( r= 0.31, p-value 0.01) in case group. Conclusion: The results of present study showed that serum levels of cortisol were significantly increased in hypothyroidism patients as compared to normal healthy controls.

Heterogeneous natural history of Addison's disease: mineralocorticoid deficiency may predominate.

Autoimmune Addison's disease (AAD) is defined as primary adrenal insufficiency due to immune-mediated destruction of the adrenal cortex. This destruction of steroid-producing cells has historically been thought of as an irreversible process, with linear progression from an ACTH-driven compensated phase to overt adrenal insufficiency requiring lifelong glucocorticoid replacement. However, a growing body of evidence suggests that this process may be more heterogeneous than previously thought, with potential for complete or partial recovery of glucocorticoid secretion. Although patients with persistent mineralocorticoid deficiency despite preserved or recovered glucocorticoid function are anecdotally mentioned, few well-documented cases have been reported to date. We present three patients in the United Kingdom who further challenge the long-standing hypothesis that AAD is a progressive, irreversible disease process. We describe one patient with a 4-year history of mineralocorticoid-only Addison's disease, a patient with spontaneous recovery of adrenal function and one patient with clinical features of adrenal insufficiency despite significant residual cortisol function. All three patients show varying degrees of mineralocorticoid deficiency, suggesting that recovery of zona fasciculata function in the adrenal cortex may occur independently to that of the zona glomerulosa. We outline the current evidence for heterogeneity in the natural history of AAD and discuss possible mechanisms for the recovery of adrenal function.

Relationship between Ca2+ and cAMP as second messengers in ACTH-induced cortisol production in bovine adrenal fasciculata cells.

In adrenal fasciculata cells stimulated by ACTH, Ca2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca2+-containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca2+-free medium. ACTH rapidly increased the intracellular free Ca2+ concentration ([Ca2+]i) in the normal medium, but did not influence [Ca2+]i in the Ca2+-free medium, indicating that ACTH caused Ca2+ influx into the cells. ACTH-induced Ca2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca2+ channel blocker but not by a T-type, N-type, or P-type Ca2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca2+-free medium and slowly caused Ca2+ influx into the cells. These results strongly suggest that Ca2+, as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

Ectopic localization of CYP11B1 and CYP11B2-expressing cells in the normal human adrenal gland.

The sharp line of demarcation between zona glomerulosa (ZG) and zona fasciculata (ZF) has been recently challenged suggesting that this interface is no longer a compartment boundary. We have used immunohistochemical analyses to study the steroid 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) pattern of expression and investigate the remodeling of the adrenal cortex in relation to aging. We analyzed human adrenal glands prepared from 47 kidney donors. No aldosterone-producing micronodules (APMs) were detectable in the younger donors aged between 22-39 but the functional ZG depicted by positive CYP11B2 staining demonstrated a lack of continuity. In contrast, the development of APMs was found in samples from individuals aged 40-70. Importantly, the progressive replacement of CYP11B2-expressing cells in the histological ZG by CYP11B1-expressing cells highlights the remodeling capacity of the adrenal cortex. In 70% of our samples, immunofluorescence studies revealed the presence of isolated or clusters of CYP11B2 positive cells in the ZF and zona reticularis. Our data emphasize that mineralocorticoid- and glucocorticoid-producing cells are distributed throughout the cortex and the medulla making the determination of the functional status of a cell or group of cells a unique tool in deciphering the changes occurring in adrenal gland particularly during aging. They also suggest that, in humans, steroidogenic cell phenotype defined by function is a stable feature and thus, the functional zonation might be not solely maintained by cell lineage conversion/migration.