11β-hydroxysteroid Dehydrogenase Research Articles

Petunidin attenuates vinclozolin instigated testicular toxicity in albino rats via regulating TLR4/MyD88/TRAF6 and Nrf-2/Keap-1 pathway: A pharmacodynamic and molecular simulation approach

Vinclozolin (VZN) is a widely used fungicide which exerts deleterious impacts on various organs including testis. Petunidin (PDN) is a polyphenolic compound that demonstrates a broad range of pharmacological activities. Thirty-two rats were divided into 4 groups including the control, VZN (100 mg/kg), VZN (100 mg/kg) + PDN (4 mg/kg) and PDN (4 mg/kg) treated group. The activities of antioxidant enzymes were assessed by using previously documented protocols. The gene expressions were determined by using qRT-PCR. The levels of hepatic function and apoptotic markers were evaluated by using standard ELISA technique. The histological analysis was carried out as per the standard protocol of histology. It was revealed that VZN disrupted the Nrf-2/Keap-1 pathway. Moreover, the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), heme-oxygenase-1 (HO-1) and glutathione reductase (GSR) were reduced whereas levels of reactive oxygen species (ROS) & malondialdehyde (MDA) were promoted following the VZN intoxication. Furthermore, VZN intoxication reduced total sperm count, viability, motility as well as luteinizing hormone (LH), follicle stimulating hormone (FSH), and plasma testosterone. Besides, administration of VZN decreased the expressions of 3β-Hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR) and 17β-Hydroxysteroid dehydrogenase (17β-HSD). Moreover, VZN exposure escalated the expressions of Bcl-2–associated X protein (Bax) and cysteine–aspartic acid protease-3 (Caspase-3) while reducing the expressions of B-cell lymphoma-2 (Bcl-2). Additionally, VZN administration increased the gene expression of toll-like receptor 4 (TLR4), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and myeloid differentiation primary response 88 (MyD88). The levels of interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the activity of cyclooxygenase-2 (COX-2) were promoted following the VZN administration. Furthermore, VZN intoxication disrupted the normal histology of testicular tissues. However, VZN + PDN treatment ameliorated testicular damage via regulating aforementioned dysregulations owing to its anti-inflammatory, antioxidative as well as anti-apoptotic potentials. Lastly, molecular docking (MD) was performed to assess the effectiveness of PDN as a curative compound by analyzing its binding affinity with the targeted proteins (Keap1, TLR4 and StAR). Our in-silico evaluations confirmed that PDN possesses the potential to interact with binding pockets of these proteins, emphasizing its capability as a curative compound to mitigate VZN-prompted reproductive damage.

Glycyrrhizic acid and patchouli alcohol in Huoxiang Zhengqi attenuate intestinal inflammation and barrier injury via regulating endogenous corticosterone metabolism mediated by 11β-HSD1

Ethnopharmacological relevanceUlcerative colitis (UC), a chronic inflammatory bowel disease, has become a significant public health challenge due to the limited effectiveness of available therapies. Huoxiang Zhengqi (HXZQ), a well-established traditional Chinese formula, shows potential in managing UC, as suggested by clinical and pharmacological studies. However, the active components and mechanisms responsible for its effects remain unclear. Aim of studyThis study aimed to identify the bioactive components of HXZQ responsible for its therapeutic effects on UC and to elucidate their underlying mechanisms. Materials and methodsThe effect of HXZQ against dextran sodium sulfate (DSS) -induced colitis was investigated. Ingredients in HXZQ were characterized and analyzed in colitic mice using liquid chromatography–mass spectrometry (LC-MS) and gas chromatography–mass spectrometry (GC-MS). In vitro, biological activity of compounds was assessed using lipopolysaccharide (LPS)-induced Ana-1 cells and bone marrow-derived macrophages (BMDMs), tumor necrosis factor-alpha-induced Caco-2 cells, and isolated intestinal crypts from colitic mice. These results were confirmed in vivo. The targets of the components were identified through bioinformatics analysis and validated via molecular docking, enzyme inhibition assays, and in vivo experiments. Hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative real-time polymerase chain reaction (qPCR) were employed to confirm the pharmaceutical effects. ResultsA clinical equivalent dose of HXZQ (2.5 mL/kg) effectively treated DSS-induced colitis. 113 compounds were identified in HXZQ, with 35 compounds detected in colitic mice. Glycyrrhizic acid (GA) and patchouli alcohol (PA) emerged as key contributors to the anti-colitic effects of HXZQ. Further investigation revealed that HXZQ and its active components decreased the levels of pro-inflammatory cytokines TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6) in colon, likely by inhibiting nuclear factor kappa-B (NF-κB) signaling pathway. This inhibition indirectly activated the intestinal farnesoid X receptor (FXR) signaling pathway, correcting bile acid imbalances caused by colitis. Additionally, these components significantly enhanced the expression of tight junction proteins ZO-1 and Occludin, as well as the adhesion protein E-cadherin, and reduced goblet cell loss, thereby repairing intestinal barrier injury. Mechanistically, GA and PA were found to inhibit 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activity, leading to increased local active corticosterone levels in the intestine to exert anti-inflammatory effects. Notably, the inhibition of 11β-HSD1 with the selective inhibitor BVT ameliorated colitis in mice. ConclusionsHXZQ exhibits therapeutic effects on UC, primarily through GA and PA inhibiting 11β-HSD1. This suggests new natural therapy approaches for UC and positions 11β-HSD1 as a potential target for colitis treatment.

α-Linolenic acid promotes testosterone synthesis by improving mitochondrial function in primary rooster Leydig cells

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. MethodsPurified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. ResultsOur results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). ConclusionALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

Oleanolic acid purified from the stem bark of Olax subscorpioidea Oliv. inhibits the function and catalysis of human 17β-hydroxysteroid dehydrogenase 1

Cancer is a leading cause of global death. Medicinal plants have gained increasing attention in cancer drug discovery. In this study, the stem bark extract of Olax subscorpioidea, which is used in ethnomedicine to treat cancer, was subjected to phytochemical investigation leading to the isolation of oleanolic acid (OA). The structure was elucidated by 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopic (NMR) data, and by comparing its data with previously reported data. Molecular docking was used to investigate the interactions of OA with nine selected cancer-related protein targets. OA docked well with human 17β-hydroxysteroid dehydrogenase type-1 (17βHSD1), caspase-3, and epidermal growth factor receptor tyrosine kinase (binding affinities: −9.8, −9.3, and −9.1 kcal/mol, respectively). OA is a triterpenoid compound with structural similarity to steroids. This similarity with the substrates of 17βHSD1 gives the inhibitor candidate an excellent opportunity to bind to 17βHSD1. The structural and functional dynamics of OA-17βHSD1 were investigated by molecular dynamics simulations at 240 ns. Molecular mechanics/Poisson-Boltzmann surface area (MMPBSA) studies showed that OA had a binding free energy that is comparable with that of vincristine (–52.76, and −63.56 kcal/mol, respectively). The average C-α root mean square of deviation (RMSD) value of OA (1.69 Å) compared with the unbound protein (2.01 Å) indicated its high stability at the protein’s active site. The binding energy and stability at the active site of 17βHSD1 recorded in this study indicate that OA exhibited profound inhibitory potential. OA could be a good scaffold for developing new anti-breast cancer drugs.

Promising Directions for Regulating Signaling Pathways Involved in the Type 2 Diabetes Mellitus Development

Many studies confirm that substances of natural origin have a pronounced affinity for type 2 diabetes mellitus (T2DM) therapeutic targets. At the moment, there is growing interest in bioactive peptides, phytochemicals, and drugs from other natural sources as highly effective, safe and promising antidiabetic agents. Natural sources are a promising resource for regulating several pathological pathways in T2DM. The review describes ways to mitigate insulin resistance and tissue sensitivity to glucose through PTP1β (protein tyrosine phosphatase 1β), GLP-1R (glucagon-like peptide receptor), DPP-4 (dipeptidyl peptidase-4), AMPK (adenosine monophosphate activated protein kinase), MAPK (mitogen-activated protein kinase). Regulation of obesity and oxidative stress development through CCN3 (nephroblastoma overexpressed gene), PPAR-γ (peroxisome proliferator-activated receptor γ), Nrf2 (nuclear factor erythroid-related factor 2), FFAR (free fatty acid receptors), 11β-HSD1 (11β-hydroxysteroid dehydrogenase). Regulation of hyperglycemia through alpha-amylase inhibitors, regulation of glucose metabolism through GFAT (glutamine fructose-6-phosphate aminotransferase), FOXO1 (forkhead box protein O1), GLUT4 (glucose transporter type 4), PGC-1α (receptor gamma coactivator 1α activating peroxisome proliferator). The review examines the use of natural sources, from which low-molecular-weight and peptide compounds are used as T2DM targets modulators.

Dysregulation of the 3β-hydroxysteroid dehydrogenase type 2 enzyme and steroid hormone biosynthesis in chronic kidney disease

IntroductionChronic kidney disease (CKD) presents a critical global health challenge, marked by the progressive decline of renal function. This study explores the role of the 3β-hydroxysteroid dehydrogenase type 2 enzyme (HSD3B2) and the steroid hormone biosynthesis pathway in CKD pathogenesis and progression.MethodsUsing an adenine-induced CKD mouse model, we conducted an untargeted metabolomic analysis of plasma samples to identify key metabolite alterations associated with CKD. Immunohistochemistry, Western blotting, and qPCR analyses were performed to confirm HSD3B2 expression in both human and mouse tissues. Additionally, Nephroseq and Human Protein Atlas data were utilized to assess the correlation between HSD3B2 and kidney function. Functional studies were conducted on HK2 cells with HSD3B2 knockdown to evaluate the impact on cell proliferation and apoptosis.ResultsMetabolic characteristics revealed significant shifts in CKD, with 61 metabolites increased and 65 metabolites decreased, highlighting the disruption in steroid hormone biosynthesis pathways influenced by HSD3B2. A detailed examination of seven key metabolites underscored the enzyme's central role. HSD3B2 exhibited a strong correlation with kidney function, supported by data from Nephroseq and the Human Protein Atlas. Immunohistochemistry, Western blotting, and qPCR analyses confirmed a drastic reduction in HSD3B2 expression in CKD-affected kidneys. Suppressed proliferation and increased apoptosis rates in HSD3B2 knocked down HK2 cells further demonstrated the enzyme's significance in regulating renal pathophysiology.DiscussionThese findings underscore the potential of HSD3B2 as a clinical diagnostic and therapeutic target in CKD. While further studies are warranted to fully elucidate the mechanisms, our results provide valuable insights into the intricate interplay between steroid hormone biosynthesis and CKD. This offers a promising avenue for precision medicine approaches and personalized treatment strategies.

Pancreatic inflammation induced by hypothyroidism in female rabbits is associated with cholesterol accumulation and a reduced expression of CYP51A1, FXRβ, and PPARβ/δ.

In women and animal models, hypothyroidism induces hypercholesterolemia, pancreatitis, and insulitis. We investigated whether lipids are involved in the effects of hypothyroidism in the pancreas. Control (n = 6) and hypothyroid (n = 6) adult female rabbits were used. We quantified serum and pancreatic triacylglycerol and total cholesterol levels, the oxidative and antioxidant status, and the expression of low-density lipoprotein cholesterol receptor (LDLR) in the pancreas. Inflammation of the pancreas was evaluated by infiltration of immune cells positive to CD163 and α-farnesoid receptor (FXRα). Other lipid players involved in both inflammation and insulin secretion of the pancreas, such as lanosterol 14-α-demethylase (CYP51A1), β-farnesoid receptor (FXRβ), 3β-hydroxysteroid dehydrogenase (3β-HSD), and peroxisome proliferator-activated receptor β (PPARβ/δ), were quantified. Groups were compared by t-Student or U-Mann-Whitney tests (p ≤ 0.05). Hypothyroidism induced hypercholesterolemia and a high cholesterol accumulation in the pancreas of female rabbits, without affecting oxidative or antioxidative status nor the expression of LDLR. The pancreas of hypothyroid females showed inflammation identified by a great infiltration of immune cells, macrophages CD163+, and loss of expression of FXRα+ in immune cells. Moreover, a reduced expression of CYP51A1, FXRβ, and PPARβ/δ, but not 3β-HSD, in the hypothyroid pancreas was found. Pancreatitis and insulitis promoted by hypothyroidism may be related to the accumulation of cholesterol, lanosterol actions, and the activation of PPARβ/δ. All inflammatory markers evaluated in this study are related to glucose regulation, suggesting the link between hypothyroidism and diabetes.

Impaired 11β-hydroxysteroid dehydrogenase type 2 activity in kidney disease disrupts 11-oxygenated androgen biosynthesis.

11-oxygenated androgens are a group of adrenal-derived steroids that require peripheral activation. In vitro data highlight a putative role for 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) in 11-oxygenated androgen biosynthesis, converting 11β-hydroxyandrostenedione (11OHA4) to 11-ketoandrostenedione (11KA4), the direct precursor of the potent androgen 11-ketotestosterone (11KT). As the kidney is the major site of HSD11B2 expression, we hypothesized that patients with chronic kidney disease (CKD) would have reduced 11-oxygenated androgen biosynthesis due to impaired HSD11B2 activity. To determine the role of HSD11B2 in 11-oxygenated androgen biosynthesis using a human CKD cohort alongside complementary cell culture and computational modeling approaches. Cross-sectional observational study of patients with CKD (n=85) and healthy controls (n=46) measuring serum and urinary concentrations of glucocorticoids, classic and 11-oxygenated androgens by liquid chromatography-tandem mass spectrometry. A computational model of peripheral 11-oxygenated androgen biosynthesis was fitted to the serum data to calculate relative HSD11B2 expression levels for each participant. HSD11B2 activity declined with eGFR, evidenced by higher cortisol (F)/cortisone (E) ratios in CKD patients compared to controls (p<0.0001). Serum concentrations of E, 11KA4, 11KT and 11β-hydroxytestosterone were lower in patients with CKD compared to controls (p<0.0001 for each). A computational model based on enzyme kinetic parameters of HSD11B2, 11β-hydroxysteroid dehydrogenase type 1, 17β-hydroxysteroid dehydrogenase type 2 and aldo-keto reductase 1C3 confirmed HSD11B2 as the key enzyme responsible for reduced 11-oxygenated androgen biosynthesis in CKD. Predicted HSD11B2 expression correlated with eGFR. This is the first in vivo study to confirm a central role for renal HSD11B2 in 11-oxygenated androgen biosynthesis. Determining the clinical implications of this observation for patients with CKD requires further research.

Pre-Conception Androgen Levels and Obstetric Outcomes in Polycystic Ovary Syndrome: A Single-Center Retrospective Study

Hyperandrogenism is a determining diagnostic factor for PCOS. If pregnancy is conceived, it is considered high-risk due to several potential complications, but the correlation between pre-pregnancy androgen levels and obstetric outcomes is poorly characterized. Objective: To determine if pre-pregnancy serum androgen concentrations and androgen indexes differed when certain obstetric and neonatal outcomes appeared in PCOS. Methods: A single-center, retrospective study was carried out. All patients were treated between 2012 and 2019. A total of 73 patients had all the endocrine and obstetric data available. Pre-pregnancy hormone levels (total testosterone-T, androstenedione-AD, DHEAS (dehydroepiandrosterone sulfate), SHBG (sex-hormone-binding globulin), and TSH (thyroid-stimulating hormone) were collected, and T/SHBG, AD/SHBG, DHEAS/SHBG, T/AD indexes were calculated and compared. Results: When miscarriage was present in the history, significantly elevated pre-pregnancy AD levels were observed. Higher pre-pregnancy AD level was noted in PCOS patients delivering female newborns as compared to males. Additionally, a higher T/AD ratio was associated with subsequent preterm delivery, but significance was lost after age adjustment. Maternal age at delivery had a significant negative correlation with pre-pregnancy DHEAS levels and DHEAS/SHBG ratio. Pre-pregnancy SHBG displayed a significant negative correlation, while pre-pregnancy androgen/SHBG ratios exhibited positive correlations with both birth weight and birth weight percentile. Conclusions: Based on our data, AD and the T/AD ratio emerge as distinctive factors in certain outcomes, implying a potential specific role of altered 17-β-HSD (17β-hydroxysteroid dehydrogenase) enzyme activity, possibly influencing offspring outcomes. The pre-pregnancy T/SHBG ratio exhibits a potentially stronger correlation with fetal growth potential compared to SHBG alone. DHEAS and maternal age at delivery are strongly correlated in PCOS patients.

7274 Assessing The Interactome Of Hexose-6-Phosphate Dehydrogenase In Breast Cancer Cells And Identifying Anterior Gradient Protein 2 As Interacting Partner By Using A BioID Method

Abstract Disclosure: G. Sakalauskaite: None. M. Weingartner: None. T. Bock: None. J. Birk: None. M. Tsachaki: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway in the endoplasmic reticulum (ER). It is so far the only enzyme shown to generate NADPH in the ER. H6PD is involved in physiological processes such as glucose and steroid metabolism, redox regulation, and response to ER-stress. So far, only one H6PD interacting partner, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), has been reported. This enzyme uses NADPH for its well-reported role in glucocorticoid activation. However, H6PD has 11β-HSD1-independent functions, such as the recently described modulation of breast cancer cell properties. Here, we adapted the BioID approach to identify novel H6PD interacting partners in the ER. First, we generated a triple negative breast cancer cell line MDA-MB 231 stably expressing a H6PD-biotin ligase fusion protein. Then, we validated the method to confirm the known interaction between H6PD and 11β-HSD1. Next, we analyzed enriched biotinylated proteins by mass spectrometry and further assessed potential candidates by co-immunoprecipitation and functional assays. The resulting interactome revealed a potential hit cluster of protein disulfide isomerase (PDI) family members, different ER-resident chaperones and luminal calcium binding proteins. Due to its association with breast cancer, we further examined the PDI Anterior gradient protein 2 (AGR2) as H6PD interacting partner. Physical interaction between AGR2 and H6PD was confirmed by co-immunoprecipitation assay in MCF7 cells. Downregulation of AGR2 by siRNA resulted in elevated H6PD protein levels but decreased activity. Furthermore, increased H6PD activity was observed when this protein was co-expressed with AGR2 in HEK-293 cells. In conclusion, the results validated the use of the BioID approach to identify protein interactions within the ER and they revealed that AGR2 physically interacts with H6PD, thereby controlling its expression and enzyme activity. Presentation: 6/3/2024

Synthesis of Carborane–Thiazole Conjugates as Tyrosinase and 11β-Hydroxysteroid Dehydrogenase Inhibitors: Antiproliferative Activity and Molecular Docking Studies

The presented study depicts the synthesis of 11 carborane–thiazole conjugates with anticancer activity, as well as an evaluation of their biological activity as inhibitors of two enzymes: tyrosinase and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The overexpression of tyrosinase results in the intracellular accumulation of melanin and can be observed in melanoma. The overexpression of 11β-HSD1 results in an elevation of glucocorticoid levels and has been associated with the aggravation of metabolic disorders such as type II diabetes mellitus and obesity. Recently, as the comorbidity of melanomas and metabolic disorders is being recognized as an important issue, the search for new therapeutic options has intensified. This study demonstrates that carborane–thiazole derivatives inhibit both enzymes, exerting beneficial effects. The antiproliferative action of all newly synthesized compounds was evaluated using three cancer cell lines, namely A172 (human brain glioblastoma), B16F10 (murine melanoma) and MDA-MB-231 (human breast adenocarcinoma), as well as a healthy control cell line of HUVEC (human umbilical vein endothelial cells). The results show that 9 out of 11 newly synthesized compounds demonstrated similar antiproliferative action against the B16F10 cell line to the reference drug, and three of these compounds surpassed it. To the best of our knowledge, this study is the first to demonstrate dual inhibitory action of carborane–thiazole derivatives against both tyrosinase and 11β-HSD1. Therefore, it represents the first step towards the simultaneous treatment of melanoma and comorbid diseases such as type II diabetes mellitus.

7285 Inhibition of 11β-Hydroxysteroid Dehydrogenase 2 by a Gut Microbiome Derived Metabolite

Abstract Disclosure: S.O. Moser: None. M.D. McCurry: None. J. Walsh: None. D.V. Winter: None. M. Smieško: None. D.J. Morris: None. A.S. Devlin: None. A. Odermatt: None. In sodium-transporting epithelial cells, 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) protects the mineralocorticoid receptor (MR) from activation by glucocorticoids. MR activation results in sodium and water retention and potassium excretion. Apparent mineralocorticoid excess (AME) results from HSD11B2 mutations, leading to cortisol-driven MR activation characterized by low renin and aldosterone levels. 11βHSD2 inhibition contributes to acquired AME, associated with essential hypertension. Licorice-derived glycyrrhetinic acid inhibits 11βHSD2, allowing cortisol accumulation and subsequent MR activation. A "cloud" of steroids and steroid-like factors is proposed to influence the activities of 11βHSD isoforms and modulate MR and GR activity in vivo. These endogenous steroidal compounds, termed glycyrrhetinic acid-like factors (GALFs), are produced by peripheral tissues and gut microbiota. GALFs exhibit potent inhibitory effects towards 11βHSD2. The significance of these GALFs in the microenvironment is emphasized when investigating essential hypertension. Experimental interventions, such as antibiotic treatment and fecal transplant, demonstrated the microbiota's influence on blood pressure regulation. 3α,11β-Dihydroxy-5β-tetrahydroprogesterone (3α,11β-diOH-5β-THP) and 3β,11β-dihydroxy-5α-tetrahydroprogesterone (3β,11β-diOH-5α-THP) are metabolites that may be formed from tetrahydrocorticosterone by the gut microbiota. In this study, we assessed the potential of 3α,11β-diOH-5β-THP and 3β,11β-diOH-5α-THP to inhibit 11βHSD2 activity as well as their capability to modulate MR transcriptional activity. Comparison of 3α,11β-diOH-5β-THP and 3β,11β-diOH-5α-THP showed that 3β,11β-diOH-5α-THP is a potent inhibitor of human 11βHSD2, with an IC50 of 47 nM whilst 3α,11β-diOH-5β-THP had an IC50 of approximately 10 µM. Both metabolites were less potent inhibitors against mouse 11βHSD2, with an IC50 of 1.3 µM for 3β,11β-diOH-5α-THP and about 25% inhibition at 20 µM 3α,11β-diOH-5β-THP. 3α,11β-diOH-5α-THP was previously shown to inhibit 11βHSD2 with an IC50 of 0.12 µM using sheep microsomes. This data suggests that a 5α-conformation of the steroid core is important for the inhibitory capacity towards 11βHSD2. 3β,11β-diOH-5α-THP was able to activate human MR, although with an EC50 two or three orders of magnitude higher than those for cortisol or aldosterone. Similarly, 3β,11β-diOH-5α-THP was able to activate mouse MR, with an one or two orders in magnitude higher EC50 than those for cortisol and aldosterone. 3α,11β-diOH-5β-THP did not activate human and mouse MR at 1 µM. In conclusion, these results highlight an endogenous metabolite that influences the functional interaction of 11βHSD2 and MR, with relevance for essential hypertension. Presentation: 6/2/2024

7963 Analysis of the inhibition of adrenal synthesis and peripheral metabolism of corticosteroids by oteseconazole and two preclinical tetrazole antifungals

Abstract Disclosure: M. Jäger: None. V. González-Ruiz: None. F.L. Joos: None. D.V. Winter: None. J. Boccard: None. T. Degenhardt: Employee; Self; Mycovia Inc. S. Brand: Employee; Self; Mycovia Inc.. S. Rudaz: None. G.R. Thompson: None. A. Odermatt: None. The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia by blocking adrenal cortisol synthesis and its peripheral inactivation [1]. Mechanisms of pseudohyperaldosteronism include inhibition of adrenal cytochrome P450 enzymes CYP11B1 and CYP17A1, as well as peripherally expressed 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). To enhance specificity for fungal CYP51, tetrazole compounds have been developed [2]. Here, we used adrenal H295R cells and enzyme activity assays to assess the inhibition of steroidogenesis by the tetrazoles oteseconazole, VT-1129 and VT-1598. In addition, the three tetrazoles were tested for inhibition of 11β-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted LC-HRMS with steroid annotation indicated common features of the oteseconazole and VT-1129 patterns with that of the triazole itraconazole, and similarities between VT-1598 and the triazole isavuconazole. Targeted quantification of nine adrenal steroids by UHPLC-MS/MS in supernatants of treated H295R cells showed an overall inhibition of steroidogenesis by the tetrazoles and the triazoles itraconazole and isavuconazole. Experiments using recombinant human enzymes suggested that this effect is not caused by direct inhibition of steroidogenic enzymes because no or very weak inhibition could be observed. Furthermore, oteseconazole, VT-1129 and VT-1598 did not inhibit 11β-HSD2, suggesting that they unlikely cause pseudohyperaldosteronism. Further research needs to uncover the exact mechanism for the observed overall inhibition of steroidogenesis by the tetrazoles as well as itraconazole and isavuconazole, and whether concentrations reached in patients are high enough to cause adrenal insufficiency and hyperplasia.

12434 History Of Surgical Menopause Is Associated With An Accelerated “Adrenal Aging” In Menopausal Women

Abstract Disclosure: J. Saini: None. R. Nathani: None. J. Larson: None. V. Fell: None. I. Bancos: None. Background: Surgical menopause due to bilateral salpingo-oophorectomy (BSO) at a younger age is associated with an increased risk of cardiovascular disease, cognitive decline, and increased mortality. Aging is associated with abnormalities in steroid metabolome, most notably an increased glucocorticoid to androgen ratio (“adrenal aging”). We hypothesized that in postmenopausal women of the same age, the duration of menopause is associated with adrenal aging. Objective: To determine the differences in the adrenal steroid metabolome of women with a history of BSO as compared to age-matched women following natural menopause. Methods: We conducted a single-center cross-sectional study in postmenopausal women with or without a history of BSO, between December 2020 to December 2022 Each participant collected 24-hour urine, which was analyzed for steroid profiling by liquid chromatography-tandem mass spectrometry (25 steroids). Exclusion criteria were inability to collect a 24h urine sample, exogenous glucocorticoid use, advanced malignancy, and chronic opioid use. Primary endpoints were glucocorticoid/androgen ratio, 11β-hydroxysteroid dehydrogenase type 2 activity (cortisol/cortisone, Tetrahydrocortisol (THF) + 5 α-tetrahydrocortisol (5 α-THF)/ Tetrahydrocortisone, THE) and 5 α-reductase type activity (Tetrahydrocortisol (THF)/5 α-tetrahydrocortisol (5 α-THF) and Etiocholanolone/Androsterone, Et/An). Results: A total of 245 women were enrolled: 124 women (median age of 66 years, IQR 63-72) with a history of BSO and 121 women (median age of 68 years, IQR 64-71) who underwent natural menopause (P=0.214). Median age at menopause was 44 years (IQR 41-46) in the BSO group, and 50 years (IQR 48-53) in the natural menopause group. History of hormone replacement therapy with estrogen was reported in 161 (66%) women, more frequently (92% vs 39%, P&amp;lt;0.001) and for a longer duration (median 12 years (IQR 6-16) vs 6 years (IQR 3-9), P&amp;lt;0.001) in in the BSO group, as compared to the natural menopause group. Although both groups had similar levels of urinary androgens (median 1387 vs 1686 mcg/24h, P=0.063) and total glucocorticoids (median 10877 vs 10277 mcg/24h, P=0.354), women with a history of BSO demonstrated a higher glucocorticoid/androgen ratio (median 7.9 (IQR 5.0-11.1) vs 6.7 (4.3-9.9), P=0.014), and a lower Et/An ratio (median 1.4 vs 1.6, P=0.045), but not THF/5aTHF ratio (median 1.7 vs 1.8, P=0.380) in comparison to women with natural menopause. No differences were found in other ratios. Conclusion: Women with a history of BSO demonstrated a higher glucocorticoid/androgen ratio in comparison to women undergoing natural menopause, suggestive of BSO-induced accelerated “adrenal aging”. Future studies need to examine associations between the abnormal steroid metabolome and increased cardiovascular morbidity, frailty, and mortality reported following BSO. Presentation: 6/3/2024

Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

Excessive glucocorticoid (GC) action is linked to various metabolic disorders. Recent findings suggest that disrupting skeletal GC signaling prevents bone loss and alleviates metabolic disorders in high-fat diet (HFD)-fed obese mice, underpinning the neglected contribution of skeletal GC action to obesity and related bone loss. Here, we show that the elevated expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), the enzyme driving local GC activation, and GC signaling in osteoblasts, are associated with bone loss and obesity in HFD-fed male mice. Osteoblast-specific 11β-HSD1 knockout male mice exhibit resistance to HFD-induced bone loss and metabolic disorders. Mechanistically, elevated 11β-HSD1 restrains glucose uptake and osteogenic activity in osteoblast. Pharmacologically inhibiting osteoblastic 11β-HSD1 by using bone-targeted 11β-HSD1 inhibitor markedly promotes bone formation, ameliorates glucose handling and mitigated obesity in HFD-fed male mice. Taken together, our study demonstrates that osteoblastic 11β-HSD1 directly contributes to HFD-induced bone loss, glucose handling impairment and obesity.

Evaluation of 3β-hydroxysteroid dehydrogenase activity using progesterone and androgen receptors-mediated transactivation.

3β-Hydroxysteroid dehydrogenases (3β-HSDs) catalyze the oxidative conversion of delta (5)-ene-3-beta-hydroxy steroids and ketosteroids. Human 3β-HSD type 2 (HSD3B2) is predominantly expressed in gonadal and adrenal steroidogenic cells for producing all classes of active steroid hormones. Mutations in HSD3B2 gene cause a rare form of congenital adrenal hyperplasia with varying degree of salt wasting and incomplete masculinization, resulting from reduced production of corticoids and androgens. Therefore, evaluation of the HSD3B2 enzymatic activity in both pathways for each steroid hormone production is important for accurately understanding and diagnosing this disorder. Using progesterone receptor (PR)- and androgen receptor (AR)-mediated transactivation, we adapted a method that easily evaluates enzymatic activity of HSD3B2 by quantifying the conversion from substrates [pregnenolone (P5) and dehydroepiandrosterone (DHEA)] to (progesterone and androstenedione). HEK293 cells were transduced to express human HSD3B2, and incubated medium containing P5 or DHEA. Depending on the incubation time with HSD3B2-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the PR/AR expression vector and progesterone-/androgen-responsive reporter. Culture media from human and other mammalian HSD3B1-expressing cells also increased the luciferase activities. HEK293 cells expressing various missense mutations in the HSD3B2 gene revealed the potential of this system to evaluate the relationship between the enzymatic activities of mutant proteins and patient phenotype.

Blockade of 11β-hydroxysteroid dehydrogenase type 1 ameliorates metabolic dysfunction-associated steatotic liver disease and fibrosis

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme involved in the conversion of cortisone to active cortisol in the liver. Elevated cortisol levels can trigger oxidative stress, inflammation, and hepatocyte damage, highlighting the importance of 11β-HSD1 inhibition as a potential therapeutic approach. This study aimed to explore the effects of INU-101, an inhibitor of 11β-HSD1, on the development of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Our findings demonstrated that INU-101 effectively mitigated cortisol-induced lipid accumulation, reactive oxygen species generation, and hepatocyte apoptosis. Furthermore, 11β-HSD1 inhibition suppressed hepatic stellate cell activation by modulating β-catenin and phosphorylated SMAD2/3. INU-101 administration significantly reduced hepatic lipid accumulation and liver fibrosis in mice fed fast-food diet. This study suggests that INU-101 holds promise as a clinical candidate for treating MASLD and fibrosis, offering potential therapeutic benefits by targeting the intricate processes involving 11β-HSD1 and cortisol regulation in the liver.

Structural determinants of parabens in inhibiting human and rat gonadal 3β-hydroxysteroid dehydrogenase

This study delved into the impacts of 10 parabens on the activity of human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) within human KGN cell and rat testicular microsomes, as well as on the secretion of progesterone in KGN cells and the inhibitory potency was compared between human and rats. Intriguingly, the outcomes revealed that ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl parabens displayed varying IC50 values for human 3β-HSD2, from 4.15 to 139.96 μM, demonstrating characteristics of mixed inhibitors. Notably, within KGN cells, all examined parabens, excluding nonyl and phenyl parabens, significantly inhibited progesterone secretion at 5–50 μM. In the case of rats, the IC50 values for these parabens on gonadal 3β-HSD1 fluctuated between 7.15 and 110.76 μM, likewise functioning as mixed inhibitors. Through docking analysis, it was proposed that most parabens effectively bind to NAD+ and/or steroid binding site. Moreover, bivariate correlation analysis unveiled an inverse correlation between IC50 values and structural characteristics such as LogP, molecular weight, heavy atom number, and carbon number within the alcohol moiety of parabens. 3D-QSAR elucidated pivotal regions, comprising hydrogen bond donor, hydrogen bond acceptor, and hydrophobic region, with the most potent inhibitor nonyl paraben engaging with all regions, while the weakest inhibitor ethyl paraben interacted with the regions except for the hydrophobic region. In conclusion, this investigation underscored the inhibitory effects imparted by several parabens on both human and rat gonadal 3β-HSD activity, with their inhibitory potency being modulated by aspects of hydrophobicity and carbon chain length.

Taste receptor T1R3 regulates testosterone synthesis via the cAMP-PKA-SP1 pathway in testicular Leydig cells

Taste receptor type 1 subunit 3 (T1R3) is a G protein-coupled receptor encoded by the TAS1R3 gene that can be specifically activated by certain sweeteners or umami agents for sweet/umami recognition. T1R3 is a potential target for regulating male reproduction. However, studies on the impact of non-nutritive sweeteners on reproduction are limited. In the present study, we evaluated the impact of the non-nutritive sweeteners (saccharin sodium, sucralose and acesulfame-K) on testosterone synthesis in testicular Leydig cells of Xiang pigs by comparing the relative abundance of mRNA transcripts and protein expression of T1R3, steroidogenic related factors, and intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), as well as testosterone levels using Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). To clarify the specific mechanism, a dual luciferase assay was used to uncover the relationship between the transcription factors and steroidogenic enzyme. The acute intratesticular injection of a typical non-nutritive sweeteners was conducted to verify this impact in mouse. The results showed that saccharin sodium not only enhanced T1R3 expression in Leydig cells of Xiang pigs, but also caused significant increases in testosterone, cAMP, PKA, phosphorylation of specificity protein 1 (p-SP1), total protein of specificity protein 1 (SP1), steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) (P<0.05). Similarly, treatment of Leydig cells with sucralose and acesulfame-K also increased testosterone level, protein expression of T1R3, 17-α-hydroxylase/17, 20-lyase (CYP17A1), and 3β-HSD1 (P<0.05). Treatment with SQ22536 (an adenylate cyclas inhibitor) or H89 (a PKA inhibitor) significantly reduced saccharin sodium-induced protein levels of p-SP1, StAR, CYP17A1, and 3β-HSD1 (P<0.05). In addition, a dual luciferase assay further demonstrated that SP1 significantly increased the promoter activity of CYP17A1 (P<0.05). When mouse testes were injected with saccharin sodium, T1R3, p-SP1, CYP17A1, and 3β-HSD1 were upregulated, leading to a significant testicular increase in testosterone and cAMP levels (P<0.05). These results suggest a mechanism by which the taste receptor T1R3 regulates testosterone production, and this mechanism may be linked to the cAMP-PKA pathway. Understanding the interrelationship between T1R3 and the cAMP-PKA-SP1 pathway contributes to clarify the regulatory mechanisms of male reproduction.

Progressive 11β-Hydroxysteroid Dehydrogenase Type 2 Insufficiency as Kidney Function Declines.

It has been postulated that chronic kidney disease (CKD) is a state of relative 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) insufficiency, resulting in increased cortisol-mediated mineralocorticoid receptor (MR) activation. We hypothesized that relative 11βHSD2 insufficiency manifests across a wide spectrum of progressively declining kidney function, including within the normal range. Adult participants were recruited at two academic centers. A discovery cohort (n=500) enrolled individuals with estimated glomerular filtration rate (eGFR) ranging from normal to CKD stage 5, in whom serum cortisol-to-cortisone (F/E) was measured as a biomarker of 11βHSD2 activity. A validation cohort (n=101) enrolled only individuals with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2) in whom 11βHSD2 activity was assessed via serum F/E and 11-hydroxy-to-11-keto androgen (11OH/K) ratios following multiple maneuvers: oral sodium suppression test (OSST), dexamethasone suppression test (DST), and ACTH-stimulation test (ACTHstim). In the discovery cohort, lower eGFR was associated with higher F/E (P-trend<0.001). Similarly, in the validation cohort, with normal eGFR, an inverse association between eGFR and both F/E and 11OH/K ratios was observed (P-trend<0.01), which persisted following DST (P-trend<0.001) and ACTHstim (P-trend< 0.05). The fractional excretion of potassium, a marker of renal MR activity, was higher with higher F/E (P-trend < 0.01) and with lower eGFR (P-trend<0.0001). A continuum of declining 11βHSD2 activity was observed with progressively lower eGFR in individuals spanning a wide spectrum of kidney function, including those with apparently normal kidney function. These findings implicate cortisol-mediated MR activation in the pathophysiology of hypertension and cardiovascular disease in CKD.