The Role of microRNA in the Regulation of Cortisol Metabolism in the Adipose Tissue in the Course of Obesity.

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.

Intratumoral cortisol associated with aromatase in the endometrial cancer microenvironment.

Glucocorticoids bind to glucocorticoid receptors (GR). In the peripheral tissues, active cortisol is produced from inactive cortisone by 11ß-hydroxysteroid dehydrogenase (HSD)1. 11ß-HSD2 is responsible for this reverse catalysis. Although GR and 11ß-HSDs have been reported to be involved in the malignant behavior of various cancer types, the concentration of glucocorticoids in cancer tissues has not been investigated. In this study, we measured glucocorticoids in serum and cancer tissues using liquid chromatography-tandem mass spectrometry and clarified, for the first time, the intratumoral "intracrine" production of cortisol by 11ß-HSD1/2 in endometrial cancer. Intratumoral cortisol levels were high in the high-malignancy type and the cancer proliferation marker Ki-67-high group, suggesting that cortisol greatly contributes to the malignant behavior of endometrial cancer. A low expression level of the metabolizing enzyme 11ß-HSD2 is more important than a high expression level of the synthase 11ß-HSD1 for intratumoral cortisol action. Intratumoral cortisol was positively related to the expression/activity of estrogen synthase aromatase, which involved GR expressed in fibroblastic stromal cells but not in cancer cells. Blockade of GR signaling by hormone therapy is expected to benefit patients with endometrial cancer.

Chemical synthesis and biochemical properties of cholestane-5α,6ß-diol-3-sulfonate: A non-hydrolysable analogue of cholestane-5α,6ß-diol-3ß-sulfate.

Cholestane-3ß,5α,6ß-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11ß-hydroxysteroid-dehydrogenase of type 2 (11ß-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6ß-diol-3ß-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6ß-diol-3ß-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11ß-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but results from the blockage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterols accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistent with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.

Tissue Glucocorticoid Metabolism in Adrenal Insufficiency: A Prospective Study of Dual-release Hydrocortisone Therapy.

BACKGROUND: Patients with adrenal insufficiency (AI) require life-long glucocorticoid (GC) replacement therapy. Within tissues, cortisol (F) availability is under the control of the isozymes of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). We hypothesize that corticosteroid metabolism is altered in patients with AI because of the nonphysiological pattern of current immediate release hydrocortisone (IR-HC) replacement therapy. The use of a once-daily dual-release hydrocortisone (DR-HC) preparation, (Plenadren®), offers a more physiological cortisol profile and may alter corticosteroid metabolism in vivo. STUDY DESIGN AND METHODS: Prospective crossover study assessing the impact of 12 weeks of DR-HC on systemic GC metabolism (urinary steroid metabolome profiling), cortisol activation in the liver (cortisone acetate challenge test), and subcutaneous adipose tissue (microdialysis, biopsy for gene expression analysis) in 51 patients with AI (primary and secondary) in comparison to IR-HC treatment and age- and BMI-matched controls. RESULTS: Patients with AI receiving IR-HC had a higher median 24-hour urinary excretion of cortisol compared with healthy controls (72.1 µg/24 hours [IQR 43.6-124.2] vs 51.9 µg/24 hours [35.5-72.3], P = .02), with lower global activity of 11ß-HSD2 and higher 5-alpha reductase activity. Following the switch from IR-HC to DR-HC therapy, there was a significant reduction in urinary cortisol and total GC metabolite excretion, which was most significant in the evening. There was an increase in 11ß-HSD2 activity. Hepatic 11ß-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11ß-HSD1 in subcutaneous adipose tissue. CONCLUSION: Using comprehensive in vivo techniques, we have demonstrated abnormalities in corticosteroid metabolism in patients with primary and secondary AI receiving IR-HC. This dysregulation of pre-receptor glucocorticoid metabolism results in enhanced glucocorticoid activation in adipose tissue, which was ameliorated by treatment with DR-HC.

Thiram, an inhibitor of 11ß-hydroxysteroid dehydrogenase type 2, enhances the inhibitory effects of hydrocortisone in the treatment of osteosarcoma through Wnt/ß-catenin pathway.

BACKGROUND: The anti-osteosarcoma effects of hydrocortisone and thiram, an inhibitor of type 2 11ß-hydroxysteroid dehydrogenase (11HSD2), have not been reported. The purpose of this study was to investigate the effects of hydrocortisone alone or the combination of hydrocortisone with thiram on osteosarcoma and the molecular mechanism, and determine whether they can be as new therapeutic agents for osteosarcoma. METHODS: Normal bone cells and osteosarcoma cells were treated with hydrocortisone or thiram alone or in combination. The cell proliferation, migration, cell cycle and apoptosis were detected by using CCK8 assay, wound healing assay, and flow cytometry, respectively. An osteosarcoma mouse model was established. The effect of drugs on osteosarcoma in vivo was assessed by measuring tumor volume. Transcriptome sequencing, bioinformatics analysis, RT-qPCR, Western blotting (WB), enzymelinked immunosorbent assay (ELISA) and siRNA transfection were performed to determine the molecular mechanisms. RESULTS: Hydrocortisone inhibited the proliferation and migration, and induced apoptosis and cell cycle arrest of osteosarcoma cells in vitro. Hydrocortisone also reduced the volume of osteosarcoma in mice in vivo. Mechanistically, hydrocortisone decreased the levels of Wnt/ß-catenin pathway-associated proteins, and induced the expression of glucocorticoid receptor α (GCR), CCAAT enhancer-binding protein ß (C/EBP-beta) and 11HSD2, resulting in a hydrocortisone resistance loop. Thiram inhibited the activity of the 11HSD2 enzyme, the combination of thiram and hydrocortisone further enhanced the inhibition of osteosarcoma through Wnt/ß-catenin pathway. CONCLUSIONS: Hydrocortisone inhibits osteosarcoma through the Wnt/ß-catenin pathway. Thiram inhibits 11HSD2 enzyme activity, reducing hydrocortisone inactivation and promoting the effect of hydrocortisone through the same pathway.

Direct inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis.

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11ß-HSD2. C8-C14 PFAS at 100 µM significantly inhibited human 11ß-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 µM) > C11 (15.09 µM) > C12 (18.43 µM) > C9 (20.93 µM) > C13 (124 µM) > C14 (147.3 µM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11ß-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11ß-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11ß-HSD2 but not rat 11ß-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11ß-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11ß-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11ß-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11ß-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11ß-HSD2.

Hepatoprotective functions of jujuboside B.

Jujuboside B (JB) found in the seeds of Zizyphi Spinosi Semen possesses pharmacological functions, such as anti-inflammatory, antiplatelet aggregation, and antianxiety potentials. This study evaluated the effect of JB on liver failure in cecal ligation and puncture (CLP)-induced sepsis. First, we observed histopathological changes in the liver by optical microscopy and the activity of enzymes in serum such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We further measured the levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, nitric oxide (NO), and antioxidative parameters in liver homogenate. The expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), and glucocorticoid receptor (GR) in the liver was observed by Western blotting. CLP enhanced the migration of inflammatory cells, ALT and AST concentrations, and necrosis, which were reduced by JB. In addition, JB reduced 11ß-HSD2 expression and levels of inflammatory mediators (TNF-α, IL-1ß, and NO) in the liver, increased GR expression, enhanced endogenous antioxidative capacity. These results further suggest that JB may protect the liver against CLP-induced damage by regulating anti-inflammatory responses, downregulating 11ß-HSD2 expression and antioxidation, and up-regulating GR expression.

Phthalates cause a low-renin phenotype commonly found in premature infants with idiopathic neonatal hypertension.

Since the 1970s, when the initial reports of neonatal hypertension related to renal artery thromboembolism were published, other secondary causes of neonatal hypertension have been reported. Those infants with no identifiable cause of hypertension were labeled with a variety of terms. Herein, we describe such infants as having idiopathic neonatal hypertension (INH). Most, but not all, of these hypertensive infants were noted to have bronchopulmonary dysplasia (BPD). More recently, reports described common clinical characteristics seen in INH patients, whether or not they had BPD. This phenotype includes low plasma renin activity, presentation near 40 weeks postmenstrual age, and a favorable response to treatment with spironolactone. A small prospective study in INH patents showed evidence of mineralocorticoid receptor activation due to inhibition of 11ß-HSD2, the enzyme that converts cortisol to the less potent mineralocorticoid-cortisone. Meanwhile, phthalate metabolites have been shown to inhibit 11ß-HSD2 in human microsomes. Premature infants can come in contact with exceptionally large phthalate exposures, especially those infants with BPD. This work describes a common low-renin phenotype, commonly seen in patients categorized as having INH. Further, we review the evidence that hypertension in INH patients with the low-renin phenotype may be mediated by phthalate-associated inhibition of 11ß-HSD2. Lastly, we review the implications of these findings regarding identification, treatment, and prevention of the low-renin hypertension phenotype seen in premature infants categorized as having INH.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

OBJECTIVE: Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. MATERIALS AND METHODS: CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11ß-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11ß-HSD2 siRNA constructs.  Expression levels of 11ß-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11ß-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. RESULTS: CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11ß-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11ß-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). CONCLUSIONS: Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11ß-HSD2 activity. We suggest that the subsequent 11ß-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Long noncoding RNA PVT1 accelerates the growth of placental trophoblasts in preeclampsia through the microRNA-24-3p/HSD11B2 axis.

Long noncoding RNA plasmacytoma variant translocation 1 (PVT1) is essential for the maintenance of normal functions of trophoblasts in preeclampsia (PE). This study aims to decipher the concrete mechanism of PVT1 with the microRNA-24-3p/Type-2 11ß-hydroxysteroid dehydrogenase (miR-24-3p/HSD11B2) axis in PE. PVT1, miR-24-3p, and HSD11B2 expression levels in normal placental tissues and PE placental tissues were defined. HTR-8/SVneo cells were transfected to determine the effects of PVT1, miR-24-3p, and HSD11B2 on the growth of HTR-8/SVneo cells. The relationships among PVT1/miR-24-3p/HSD11B2 in HTR-8/SVneo cells were identified. PVT1 and HSD11B2 were downregulated, while miR-24-3p was upregulated in the placenta of PE. Upregulated/downregulated PVT1 promoted/impeded the growth of human placental trophoblast (HTR-8/SVneo) cells in PE. Restored/knocked down miR-24-3p impeded/enhanced the growth of HTR-8/SVneo cells in PE. PVT1 inhibited miR-24-3p to mediate HSD11B2. PVT1 sponges miR-24-3p to regulate HSD11B2; thereby, the growth of placental trophoblasts is promoted in PE.

Selenium attenuates the cadmium-induced placenta glucocorticoid barrier damage by up-regulating the expression of specificity protein 1.

Cadmium (Cd) is an environmental pollutant and pregnant women are especially susceptible to the effects of exposure to Cd. Our previous study found Cd can be accumulated in the placenta and causes fetal growth restriction (FGR) through damage the placental glucocorticoid barrier. Selenium (Se), as an essential micronutrient, can allivate Cd-induced toxicity. In this study, we aim to explore the protective mechanism of Se against Cd-induced the placental glucocorticoid barrier damage and FGR. Pregnant Sprague Dawley (SD) rats were exposed to CdCl2 (1 mg/kg/day) and Na2 SeO3 (0.1-0.2-0.3 mg/kg/day) by gavage from gestational day (GD) 0 to GD 19. The results showed that reduced fetal weight, increased corticosterone concentrations in the maternal and fetal serum, and impaired placental labyrinth layer blood vessel development, appeared in pregnant rats after Cd exposure and improved after treated with Se. In cell experiments, we confirmed that Se reduces Cd-induced apoptosis. Moreover, Se can abolish Cd-induced 11ß-HSD2 and specificity protein 1 (Sp1) decreasing in vivo and vitro. In human JEG-3 cells, the knockdown of Sp1 expression by small interfering RNA can suppressed the protective effect of Se on Cd-induced 11ß-HSD2 decreasing. In general, our results demonstrated that Se is resistant to Cd-induced FGR through upregulating the placenta barrier via activation of the transcription factor Sp1.

Cadmium induces placental glucocorticoid barrier damage by suppressing the cAMP/PKA/Sp1 pathway and the protective role of taurine.

Cadmium (Cd) exposure during pregnancy damages the placental glucocorticoid (GC) barrier, exposes the foetus to excess corticosterone (CORT) levels, and eventually inhibits foetal development. In addition, taurine (Tau) alleviates the toxicity of Cd on liver and kidney, but limited data are available on the role of Tau against the toxicity of heavy metals on female reproduction and fetal development. The present study was conducted to investigate the specific mechanism of Cd-induced placental GC barrier damage and the protective role of Tau. Pregnant rats were administered CdCl2 (1 mg/kg/day) and Tau (100, 200, or 300 mg/kg/day) by gavage from gestational day (GD) 0 to 19. The data showed that CdCl2 increased the foetal growth restriction (FGR) rate of the offspring, and the levels of CORT in the placental, maternal and foetal serum. Treatment with Tau significantly reversed the impact of Cd on both maternal and fetal parameters. Additionally, Tau can attenuate Cd-induced inhibition of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) and specificity protein 1 (Sp1) in vivo and vitro. Furthermore, Sp1-siRNA alone reduced 11ß-HSD2 levels and had a further inhibitory effect when the cells were treated with Cd simultaneously. Moreover, Cd suppressed cAMP/PKA signalling. Forskolin (adenylate cyclase agonist) pretreatment activated cAMP/PKA signalling and restored the Cd-induced downregulation of Sp1 and 11ß-HSD2. Tau alleviated the Cd-induced decrease of Sp1 via activating cAMP/PKA signalling. Therefore, the results highlight that Tau protects against Cd-induced impairments in GC barrier damage by upregulating the cAMP/PKA/Sp1 pathway in placental trophoblasts.

Glucocorticoid Production in Lymphoid Organs: Acute Effects of Lipopolysaccharide in Neonatal and Adult Mice.

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.

A life-threatening case of pseudo-aldosteronism secondary to excessive liquorice ingestion.

BACKGROUND: Liquorice is found in many food products, soft drinks, and herbal medicines. Liquorice ingestion is an uncommon cause of apparent mineralocorticoid excess or pseudo-aldosteronism. The mechanism involves the inhibition of 11-beta-hydroxysteroid dehydrogenase type-2 by the active ingredient called glycyrrhizin. This leads to the uninhibited activation of mineralocorticoid receptors by cortisol. Confectionary products that contain liquorice are readily available in many countries around the world. CASE PRESENTATION: We report a case of severe refractory hypokalaemia with hypertensive crisis and acute pulmonary oedema due to excessive liquorice consumption. A 79-year-old female presented to the emergency department following a road traffic accident. She described feeling weak and dizzy while driving before the collision. She attended her general practitioner (GP) several weeks earlier for fatigue and was being managed for hypokalaemia on oral potassium supplements. Investigations revealed hypertension (BP 180/69 mmHg), severe hypokalaemia (K 2.2 mmol/l), normal renal function, normal serum magnesium with metabolic alkalosis. Spot urinary potassium was 22 mmol/l. The patient denied taking medications including over-the-counter or herbal medication that can cause hypokalaemia. Hypokalaemia persisted despite aggressive intravenous (i.v.) and oral potassium replacement. She later developed a hypertensive crisis (BP 239/114 mmHg) with pulmonary oedema. She required admission to the intensive care unit and was managed with intravenous furosemide infusion and isosorbide dinitrate infusion. On further discussion, our patient admitted to struggling with nicotine cravings since quitting smoking two months earlier. She began eating an excessive amount of liquorice sweets to manage her cravings. Suppression of plasma renin and aldosterone supported the diagnosis of apparent mineralocorticoid excess secondary to excessive liquorice consumption. Her symptoms and hypokalaemia resolved after stopping liquorice intake. CONCLUSIONS: This case highlights the life-threatening and refractory nature of hypokalaemia secondary to excessive liquorice consumption. This case also emphasizes the importance of comprehensive history taking including dietary habits. Increased awareness among the public is required regarding the potential health hazards of excessive liquorice consumption.

A Novel N-Tert-Butyl Derivatives of Pseudothiohydantoin as Potential Target in Anti-Cancer Therapy.

Tumors are currently more and more common all over the world; hence, attempts are being made to explain the biochemical processes underlying their development. The search for new therapeutic pathways, with particular emphasis on enzymatic activity and its modulation regulating the level of glucocorticosteroids, may contribute to the development and implementation of new therapeutic options in the treatment process. Our research focuses on understanding the role of 11ß-HSD1 and 11ß-HSD2 as factors involved in the differentiation and proliferation of neoplastic cells. In this work, we obtained the 9 novel N-tert-butyl substituted 2-aminothiazol-4(5H)-one (pseudothiohydantoin) derivatives, differing in the substituents at C-5 of the thiazole ring. The inhibitory activity and selectivity of the obtained derivatives in relation to two isoforms of 11ß-HSD were evaluated. The highest inhibitory activity for 11ß-HSD1 showed compound 3h, containing the cyclohexane substituent at the 5-position of the thiazole ring in the spiro system (82.5% at a conc. 10 µM). On the other hand, the derivative 3f with the phenyl substituent at C-5 showed the highest inhibition of 11ß-HSD2 (53.57% at a conc. of 10 µM). A low selectivity in the inhibition of 11ß-HSD2 was observed but, unlike 18ß-glycyrrhetinic acid, these compounds were found to inhibit the activity of 11ß-HSD2 to a greater extent than 11ß-HSD1, which makes them attractive for further research on their anti-cancer activity.

Decreased 11ß-Hydroxysteroid Dehydrogenase Type 2 Expression in the Kidney May Contribute to Nicotine/Smoking-Induced Blood Pressure Elevation in Mice.

[Figure: see text].

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17ß-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11ß-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Smoothened-activating lipids drive resistance to CDK4/6 inhibition in Hedgehog-associated medulloblastoma cells and preclinical models.

Medulloblastoma is an aggressive pediatric brain tumor that can be driven by misactivation of the Hedgehog (HH) pathway. CDK6 is a critical effector of oncogenic HH signaling, but attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to single-agent molecular therapy. We identified mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma by performing orthogonal CRISPR and CRISPR interference screens in medulloblastoma cells treated with a CDK4/6 inhibitor and RNA-Seq of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6. Our concordant in vitro and in vivo data revealed that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to ER stress and activation of the unfolded protein response (UPR). These pathways increased the activity of enzymes producing Smoothened-activating (SMO-activating) sterol lipids that sustained oncogenic HH signaling in medulloblastoma despite cell-cycle attenuation. We consistently demonstrated that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing SMO-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. Our data reveal what we believe to be a novel pathway of resistance to CDK4/6 inhibition as well as a novel combination therapy to treat the most common malignant brain tumor in children.

Perceived stress, depressive symptoms, and cortisol-to-cortisone ratio in spot urine in 6878 older adults.

BACKGROUND: Late life depression and perceived stress could influence disease pathways via reduced 11ß-HSD2 activity, particularly given suggestions that reduced 11ß-HSD2 activity, which is reflected in the cortisol-to-cortisone ratio, is a risk factor of disease. To date, however, examination of the relationship between the cortisol-to-cortisone ratio and perceived stress or depressive symptoms is insufficient. METHODS: We examined the cross-sectional association of the cortisol-to-cortisone ratio with perceived stress and depressive symptoms, and analyzed whether cortisol levels modify this association, in 6878 participants aged 45-74 years. Cortisol and cortisone in spot urine were measured using liquid chromatography-mass spectrometry. Perceived stress during the past year was measured using a self-reported questionnaire. Depressive symptoms were evaluated using the Zung Self-Rating Depression Scale. Analyses were performed with adjustment for age, sex, lifestyle factors (smoking habit, alcohol consumption, physical activity, and sleeping hours), and physical health factors (body mass index [kg/m2] and medical history [diabetes, hypertension, and medication for hyperlipidemia or corticosteroids]). RESULTS: Cortisol-to-cortisone ratio and cortisol were positively associated with perceived stress (% change: 2.33, Ptrend = 0.003; and 4.74, Ptrend = 0.001, respectively), but were not significantly associated with depressive symptoms. Further, the relationship between cortisol-to-cortisone ratio and perceived stress was modified by cortisol level and sex: the positive association between perceived stress and the cortisol-to-cortisone ratio was more evident in subjects with lower cortisol levels (Pinteraction = 0.009) and in men (Pinteraction = 0.026). CONCLUSIONS: These findings suggest that the cortisol-to-cortisone ratio in spot urine may be a useful marker for non-acute perceived stress in daily life against a possible background of reduced 11ß-HSD2 in older adults.

Influence of labor on direct and indirect determinants of placental 11beta-hydroxysteroid dehydrogenase activity.

PURPOSE: Labor is a complex process involving multiple para-, auto- and endocrine cascades. The interaction of cortisol, corticotropin-releasing hormone (CRH) and progesterone is essential. The action of cortisol on the human feto-placental unit is regulated by 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2/HSD11B2) that converts cortisol into inactive cortisone. The majority of studies on the assessment of placental 11ß-HSD2 function determined indirect activity parameters. It remains elusive if indirect measurements correlate with enzymatic function and if these parameters are affected by potential confounders (e.g., mode of delivery). Thus, we compared determinants of indirect 11ß-HSD2 tissue activity with its direct enzymatic turnover rate in placental samples from spontaneous births and cesarean (C)-sections. METHODS: Using LC-MS/MS, we determined CRH, cortisol, cortisone, progesterone and 17-hydroxy(OH)-progesterone in human term placentas (spontaneous birth vs. C-section, n = 5 each) and measured the enzymatic glucocorticoid conversion rates in placental microsomes. Expression of HSD11B1, 2 and CRH was determined via qRT-PCR in the same samples. RESULTS: Cortisol-cortisone ratio correlated with direct microsomal enzymatic turnover. While this observation seemed independent of sampling site, a strong influence of mode of delivery on tissue steroids was observed. The mRNA expression of HSD11B2 correlated with indirect and direct cortisol turnover rates in C-section placentas only. In contrast to C-sections, CRH, cortisol and cortisone levels were significantly increased in placental samples following spontaneous birth. CONCLUSION: Labor involves a series of complex hormonal processes including activation of placental CRH and glucocorticoid metabolism. This has to be taken into account when selecting human cohorts for comparative analysis of placental steroids.