BACKGROUND: Adrenal-origin and peripheral tissue-transformed 11-oxygenated androgens are recognized as significant androgens. However, our current understanding of the synthesis of 11-oxygenated androgens, including the organs and cell types involved, remains limited.
METHODS: We performed comprehensive analyses on an extensive dataset of normal human tissues, which included bulk RNA data from 30 tissues, single-cell RNA sequencing (scRNA) data from 16 tissues and proteomics data from 29 tissues, to characterize the expression profiles of enzyme-encoding genes. To validate the findings, immunohistochemical and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques were employed.
RESULTS: Our investigation revealed that the gene expression levels of the enzymes HSD11B2 and AKR1C3 were notably elevated in the kidney and intestines. Intriguingly, within these organs, we observed an increasing trend in enzyme expression with age in women, while a decreasing trend was apparent in men. scRNA analysis revealed that HSD11B2 was predominantly expressed in collecting duct principal cells in the kidney, while AKR1C3 was primarily expressed in the proximal tubules. Intriguingly, nearly all epithelial cells in the intestine expressed these key enzymes. Further analysis using LC-MS/MS revealed that the kidney exhibited the highest levels of 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT) among the seven tissues examined, and substantial synthesis of 11KA4 and 11KT was also observed in the intestine. Finally, we developed the TransMap website (http://gxmujyzmolab.cn:16245/TransMap/) to provide comprehensive visualization of all currently available transcriptome data.
CONCLUSIONS: This study offers an overarching perspective on tracing the synthesis of 11-oxygenated androgens in peripheral tissues, thereby providing valuable insights into the potential role of these androgens in humans.

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Resveratrol is converted to various metabolites by gut microbiota. Human and rat liver 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) are critical for glucocorticoid activation, while 11β-HSD2 in the kidney does the opposite reaction. It is still uncertain whether resveratrol and its analogues selectively inhibit 11β-HSD1. In this study, the inhibitory strength, mode of action, structure-activity relationship (SAR), and docking analysis of resveratrol analogues on human, rat, and mouse 11β-HSD1 and 11β-HSD2 were performed. The inhibitory strength of these chemicals on human 11β-HSD1 was dihydropinosylvin (6.91 μM) > lunularin (45.44 μM) > pinostilbene (46.82 μM) > resveratrol (171.1 μM) > pinosylvin (193.8 μM) > others. The inhibitory strength of inhibiting rat 11β-HSD1 was pinostilbene (9.67 μM) > lunularin (17.39 μM) > dihydropinosylvin (19.83 μM) > dihydroresveratrol (23.07 μM) > dihydroxystilbene (27.84 μM) > others and dihydropinosylvin (85.09 μM) and pinostilbene (>100 μM) inhibited mouse 11β-HSD1. All chemicals did not inhibit human, rat, and mouse 11β-HSD2. It was found that dihydropinosylvin, lunularin, and pinostilbene were competitive inhibitors of human 11β-HSD1 and that pinostilbene, lunularin, dihydropinosylvin, dihydropinosylvin and dihydroxystilbene were mixed inhibitors of rat 11β-HSD1. Docking analysis showed that they bind to the steroid-binding site of human and rat 11β-HSD1. In conclusion, resveratrol and its analogues can selectively inhibit human and rat 11β-HSD1, and mouse 11β-HSD1 is insensitive to the inhibition of resveratrol analogues.

The activity of the enzyme 11β-hydroxysteroid dehydrogenase type II (11β-HSD type II), which can be estimated by the combined measurement of cortisol and cortisone, is gaining importance as a marker for the assessment of stress in pigs. The aim of this study was to investigate the activity of this enzyme and the salivary concentrations of cortisol and cortisone in pigs during pregnancy, farrowing and lactation and to compare it with other stress-related biomarkers such as Chromogranin A (CgA), S100A12 and alpha-amylase. Salivary cortisone concentrations and 11β-HSD type II activity decreased after farrowing, while cortisol concentrations increased. Enzyme activity did not show significant correlations with any of the other stress-related biomarkers measured in this study. Overall, the results of this report indicate a different regulation of 11β-HSD type II activity and of cortisol and cortisone during pregnancy and lactation, which should be considered when evaluating these analytes in saliva during these periods.

Stress-induced increases in cortisol can stimulate or inhibit brain cell proliferation, but the mechanisms behind these opposing effects are unknown. We tested the hypothesis that 11β-hydroxysteroid dehydrogenase type 2 (Hsd11b2), a glucocorticoid-inactivating enzyme expressed in neurogenic regions of the adult zebrafish brain, mitigates cortisol-induced changes to brain cell proliferation, using one of three stress regimes: a single 1 min air exposure (acute stress), two air exposures spaced 24 h apart (repeat acute stress) or social subordination (chronic stress). Plasma cortisol was significantly elevated 15 min after air exposure and recovered within 24 h after acute and repeat acute stress, whereas subordinate fish exhibited significant and sustained elevations relative to dominant fish for 24 h. Following acute stress, brain hsd11b2 transcript abundance was elevated up to 6 h after a single air exposure but was unchanged by repeat acute stress or social subordination. A sustained increase in brain Hsd11b2 protein levels occurred after acute stress, but not after repeat or chronic stress. Following acute and repeat acute stress, brain pcna transcript abundance (a marker of cell proliferation) exhibited a prolonged elevation, but was unaffected by social subordination. Interestingly, the number of telencephalic BrdU+ cells increased in fish after a single air exposure but was unchanged by repeat acute stress. Following acute and repeat acute stress, fish expressed lower brain glucocorticoid and mineralocorticoid receptor (gr and mr) transcript abundance while subordinate fish exhibited no changes. Taken together, these results demonstrate stressor-specific regulation of Hsd11b2 in the zebrafish brain that could modulate rates of cortisol catabolism contributing to observed differences in brain cell proliferation.

Pseudohyperaldosteronism (PHA) is characterized by hypertension, hypokalemia, and a decrease in plasma renin and aldosterone levels. It can be caused by several causes, but the most frequent is due to excess intake of licorice. The effect is mediated by the active metabolite of licorice, glycyrrhetinic acid (GA), which acts by blocking the 11-hydroxysteroid dehydrogenase type 2 and binding to the mineralocorticoid receptor (MR) as an agonist. The management of licorice-induced PHA depends on several individual factors, such as age, gender, comorbidities, duration and amount of licorice intake, and metabolism. The clinical picture usually reverts upon licorice withdrawal, but sometimes mineralocorticoid-like effects can be critical and persist for several weeks, requiring treatment with MR blockers and potassium supplements. Through this case series of licorice-induced PHA, we aim to increase awareness about exogenous PHA, and the possible risk associated with excess intake of licorice. An accurate history is mandatory in patients with hypertension and hypokalemia to avoid unnecessary testing. GA is a component of several products, such as candies, breath fresheners, beverages, tobacco, cosmetics, and laxatives. In recent years, the mechanisms of action of licorice and its active compounds have been better elucidated, suggesting its benefits in several clinical settings. Nevertheless, licorice should still be consumed with caution, considering that licorice-induced PHA is still an underestimated condition, and its intake should be avoided in patients with increased risk of licorice toxicity due to concomitant comorbidities or interfering drugs.

Mineralocorticoids play a key role in hydromineral balance by regulating sodium retention and potassium wasting. Through favoring sodium, mineralocorticoids can cause hypertension from fluid overload under conditions of hyperaldosteronism, such as aldosterone-secreting tumors. An often-overlooked mechanism by which aldosterone functions to increase sodium is through stimulation of salt appetite. To drive sodium intake, aldosterone targets neurons in the hindbrain which uniquely express 11β-hydroxysteroid dehydrogenase type 2 (HSD2). This enzyme is a necessary precondition for aldosterone-sensing cells as it metabolizes glucocorticoids - preventing their activation of the mineralocorticoid receptor. In this review, we will consider the role of hindbrain HSD2 neurons in regulating sodium appetite by discussing HSD2 expression in the brain, regulation of hindbrain HSD2 neuron activity, and the circuitry mediating the effects of these aldosterone-sensitive neurons. Reducing the activity of hindbrain HSD2 neurons may be a viable strategy to reduce sodium intake and cardiovascular risk, particularly for conditions of hyperaldosteronism.

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18β-glycyrrhetinic acid (18β-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Preeclampsia (PE) is a complex human-specific complication frequently associated with placental pathology. The local renin-angiotensin system (RAS) in the human placenta, which plays a crucial role in regulating placental function, has been extensively documented. Glucocorticoids (GCs) are a class of steroid hormones. PE cases often have abnormalities in GCs levels and placental GCs barrier. Despite extensive speculation, there is currently no robust evidence indicating that GCs regulate placental RAS. This study aims to investigate these potential relationships. Plasma and placental samples were collected from both normal and PE pregnancies. The levels of angiotensin-converting enzyme (ACE), angiotensin II (Ang II), cortisol, and 11β-hydroxysteroid dehydrogenases (11βHSD) were analyzed. In PE placentas, cortisol, ACE, and Ang II levels were elevated, while 11βHSD2 expression was reduced. Interestingly, a positive correlation was observed between ACE and cortisol levels in the placenta. A significant inverse correlation was found between the methylation statuses within the 11βHSD2 gene promoter and its expression, meanwhile, 11βHSD2 expression was negatively correlated with cortisol and ACE levels. In vitro experiments using placental trophoblast cells confirmed that active GCs can stimulate ACE transcription and expression through the GR pathway. Furthermore, 11βHSD2 knockdown could enhance this activating effect. An in vivo study using a rat model of intrauterine GCs overexposure during mid-to-late gestation suggested that excess GCs in utero lead to increased ACE and Ang II levels in the placenta. Collectively, this study provides the first evidence of the relationships between 11βHSD2 expression, GCs barrier, ACE, and Ang II levels in the placenta. It not only contributes to understanding the pathological features of the placental GCs barrier and RAS under PE conditions, also provides important information for revealing the pathological mechanism of PE.

The similarity of the clinical picture of metabolic syndrome and hypercortisolemia supports the hypothesis that obesity may be associated with impaired expression of genes related to cortisol action and metabolism in adipose tissue. The expression of genes encoding the glucocorticoid receptor alpha (GR), cortisol metabolizing enzymes (HSD11B1, HSD11B2, H6PDH), and adipokines, as well as selected microRNAs, was measured by real-time PCR in adipose tissue from 75 patients with obesity, 19 patients following metabolic surgery, and 25 normal-weight subjects. Cortisol levels were analyzed by LC-MS/MS in 30 pairs of tissues. The mRNA levels of all genes studied were significantly (p < 0.05) decreased in the visceral adipose tissue (VAT) of patients with obesity and normalized by weight loss. In the subcutaneous adipose tissue (SAT), GR and HSD11B2 were affected by this phenomenon. Negative correlations were observed between the mRNA levels of the investigated genes and selected miRNAs (hsa-miR-142-3p, hsa-miR-561, and hsa-miR-579). However, the observed changes did not translate into differences in tissue cortisol concentrations, although levels of this hormone in the SAT of patients with obesity correlated negatively with mRNA levels for adiponectin. In conclusion, although the expression of genes related to cortisol action and metabolism in adipose tissue is altered in obesity and miRNAs may be involved in this process, these changes do not affect tissue cortisol concentrations.