11β-HSD1 Activity Research Articles

Assessment of steroid enzymes action in children and adolescents with obesity

Rising prevalence of obesity has become an important impulse to investigate basic mechanisms involved in regulating the energy balance. It is widely accepted that steroids are potent factors affecting glucose, fat, and protein metabolism. Our study was aimed to analyze differences in the total amount of selected enzymes implicated in steroid metabolism in a group of children suffering from obesity and those with normal weight, further subdivided according to sex and pubertal stage. Data were obtained from 187 Caucasian children and adolescents, including 113 patients (63 girls, 50 boys) with obesity and 74 (34 girls, 40 boys) normal weight volunteers. Standard clinical examinations were performed in both groups. To evaluate the impact of puberty, preadolescent children and those with advanced puberty were assessed separately. Urine steroid excretion profiles were analyzed using gas chromatography/mass spectrometry method. Children with obesity revealed several changes in in the total amount of steroid enzymes as assessed by the relevant metabolite proportions, compared to their norm weight peers. Girls showed a significant increase in the activity of 11βHSD1, while boys demonstrated a relevant elevation in 20αHSD action. Regardless of sex, children with obesity showed an increase in the activity of 5β-reductase + 3αHSD complex and a decrease in the involvement of 11βOH-lase. The effect is attenuated when consider pre- and pubertal subgroups. We hypothesize that changes in the activity levels of selected enzymes may be a compensatory mechanism to limit the glucocorticoid exposure of key target tissues as well as to improve metabolic control and reduce long-term complications of obesity.

SAT278 Thyroid Hormones Regulate 11bhsd Enzymes: A Novel Function In Glucocorticoid Hormone Metabolism

Abstract Disclosure: L. Bessiene: None. J. Perrot: None. S. Hescot: None. A. Bourdin-Pintueles: None. G. Vitellius: None. L.M. Sachs: None. P. Kamenicky: None. M. Lombes: None. E. Pussard: None. S. Viengchareun: None. L. Martinerie: None. Glucocorticoid hormone metabolism is regulated by 11-beta hydroxysteroid dehydrogenase enzymes: 11βHSD2, mostly expressed in the distal nephron, converts corticosterone into 11-dehydrocorticosterone in rodents, while 11βHSD1 catalyzes the opposite reaction in liver. Glucocorticoid hormone metabolism may be altered under pathophysiological conditions of hypothyroidism. However, direct functional relationship between glucocorticoid and thyroid hormones (T3) signaling pathways remains elusive. Identification of putative Thyroid Response Elements (TRE) in the promoter regions of hsd11b2 and hsd11b1 genes suggested that T3 might directly regulate expression and/or activity of these enzymes.We used human translational studies, a mouse model of hyperthyroidism and murine renal and hepatic cell lines to investigate the role of thyroid hormones in glucocorticoid hormone metabolism.In hypothyroid patients, the urinary [E]/[F] ratio measured by LC-MS/MS technology, showed a 60% decrease (P<0.05), compared to age- and sex- matched euthyroid controls, suggesting a regulation of glucocorticoid metabolism by T3. Similarly, a significant positive correlation was observed between the urinary [E]/[F] ratio and serum T4L concentration in hyperthyroid patients before and after normalization of T4L levels.Administration of T3 in drinking water, mimicking a mouse model of hyperthyroidism, led to significant increase in renal 11βHSD2 mRNA (P<0.05) and decrease in liver 11βHSD1 mRNA and protein levels (P<0.01), compared to control mice.Finally, T3 induced an increase in 11βHSD2 transcript levels in renal KC3AC1 cells (P<0.01) and a decrease in 11βHSD1 mRNA levels in hepatic HepG2 cells (P<0.01). 11βHSD2 activity increased by 20% (P<0.05). In HEK 293T cells, T3 transactivated hsd11b2 promoter via the Thyroid Receptor α1 (TRα1). ChIP experiments further demonstrated a T3-dependent specific recruitment of TRα1 onto hsd11b2 promoter. Altogether, these findings demonstrate that T3 directly regulate expression and activity of 11βHSD enzymes, thereby controlling glucocorticoid hormone metabolism and action. Presentation: Saturday, June 17, 2023

Patulin Stimulates Progenitor Leydig Cell Proliferation but Delays Its Differentiation in Male Rats during Prepuberty.

Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.

G6PC1 and G6PC2 influence G6P flux but not HSD11B1 activity.

In the endoplasmic reticulum (ER) lumen, glucose-6-phosphatase catalytic subunit 1 and 2 (G6PC1; G6PC2) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate whereas hexose-6-phosphate dehydrogenase (H6PD) hydrolyzes G6P to 6-phosphogluconate (6PG) in a reaction that generates NADPH. 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) utilizes this NADPH to convert inactive cortisone to cortisol. HSD11B1 inhibitors improve insulin sensitivity whereas G6PC inhibitors are predicted to lower fasting blood glucose (FBG). This study investigated whether G6PC1 and G6PC2 influence G6P flux through H6PD and vice versa. Using a novel transcriptional assay that utilizes separate fusion genes to quantitate glucocorticoid and glucose signaling, we show that overexpression of H6PD and HSD11B1 in the islet-derived 832/13 cell line activated glucocorticoid-stimulated fusion gene expression. Overexpression of HSD11B1 blunted glucose-stimulated fusion gene expression independently of altered G6P flux. While overexpression of G6PC1 and G6PC2 blunted glucose-stimulated fusion gene expression, it had minimal effect on glucocorticoid-stimulated fusion gene expression. In the liver-derived HepG2 cell line, overexpression of H6PD and HSD11B1 activated glucocorticoid-stimulated fusion gene expression but overexpression of G6PC1 and G6PC2 had no effect. In rodents, HSD11B1 converts 11-dehydrocorticosterone (11-DHC) to corticosterone. Studies in wild-type and G6pc2 knockout mice treated with 11-DHC for 5 weeks reveal metabolic changes unaffected by the absence of G6PC2. These data suggest that HSD11B1 activity is not significantly affected by the presence or absence of G6PC1 or G6PC2. As such, G6PC1 and G6PC2 inhibitors are predicted to have beneficial effects by reducing FBG without causing a deleterious increase in glucocorticoid signaling.

Halogenated bisphenol A derivatives potently inhibit human, rat, and mouse gonadal 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico molecular docking analysis

Bisphenol A (BPA) is a widely used plastic material, and halogenated BPA derivatives are formed either by synthesis or environmental processes. However, the effect of halogenated bisphenols on steroidogenesis remains unclear. The aim of this study was to compare inhibition of 6 BPA derivatives on gonadal 3β-hydroxysteroid dehydrogenases (3β-HSDs) in three species (human, rat, and mouse). The inhibition on human 3β-HSD2 was tetrabromo BPA (TBBPA, IC50, 1.01 μM)>trichloro BPA (TrCBPA, 3.95 μM)>tetrachloro BPA (TCBPA, 4.14 μM)>monochloro BPA (MCBPA, 4.74 μM)>others with TrCBPA of competitive, TBBPA of noncompetitive and MCBPA/TCBPA of mixed inhibition. The inhibition on rat 3β-HSD1 was TCBPA (1.68 μM)>TrCBPA (1.72 μM)>MCBPA (2.80 μM)>BPA>others with mixed inhibition. The inhibition on mouse 3β-HSD6 was TrCBPA (1.59 μM) >MCBPA (3.36 μM)>TCBPA (3.72 μM)>others with mixed inhibition. Molecular docking analysis showed that TBBPA, TrCBPA, and TCBPA bind to steroid active sites, contacting with catalytic residue Tyr154 of human 3β-HSD2. MCBPA, TrCBPA, and TCBPA bind to steroid active site of rat 3β-HSD1. MCBPA and TrCBPA bind to active site of mouse 3β-HSD6. Regression of lowest binding energy values with Ki values revealed a significant negative linear regression (P < 0.05). In conclusion, halogenated BPA derivatives are more potent inhibitors of three 3β-HSDs than BPA and there is structure-dependent inhibition. SynopsisChlorinated bisphenol derivatives after water chlorination process and other halogenated bisphenols effectively inhibit human and rat 3β-HSD activity, thereby leading to steroid hormone deficiency.

Contribution of local regeneration of glucocorticoids to tissue steroid pools.

11β-Hydroxysteroid dehydrogenase 1 (11βHSD1) is a drug target to attenuate adverse effects of chronic glucocorticoid excess. It catalyses intracellular regeneration of active glucocorticoids in tissues including brain, liver and adipose tissue (coupled to hexose-6-phosphate dehydrogenase, H6PDH). 11βHSD1 activity in individual tissues is thought to contribute significantly to glucocorticoid levels at those sites, but its local contribution vs glucocorticoid delivery via the circulation is unknown. Here, we hypothesised that hepatic 11βHSD1 would contribute significantly to the circulating pool. This was studied in mice with Cre-mediated disruption of Hsd11b1 in liver (Alac-Cre) vs adipose tissue (aP2-Cre) or whole-body disruption of H6pdh. Regeneration of [9,12,12-2H3]-cortisol (d3F) from [9,12,12-2H3]-cortisone (d3E), measuring 11βHSD1 reductase activity was assessed at steady state following infusion of [9,11,12,12-2H4]-cortisol (d4F) in male mice. Concentrations of steroids in plasma and amounts in liver, adipose tissue and brain were measured using mass spectrometry interfaced with matrix-assisted laser desorption ionisation or liquid chromatography. Amounts of d3F were higher in liver, compared with brain and adipose tissue. Rates of appearance of d3F were ~6-fold slower in H6pdh-/- mice, showing the importance for whole-body 11βHSD1 reductase activity. Disruption of liver 11βHSD1 reduced the amounts of d3F in liver (by ~36%), without changes elsewhere. In contrast disruption of 11βHSD1 in adipose tissue reduced rates of appearance of circulating d3F (by ~67%) and also reduced regenerated of d3F in liver and brain (both by ~30%). Thus, the contribution of hepatic 11βHSD1 to circulating glucocorticoid levels and amounts in other tissues is less than that of adipose tissue.

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

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 11β-hydroxysteroid dehydrogenase (11β-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. 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. 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 11β-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 11β-HSD2 activity. Hepatic 11β-HSD1 activity was not significantly altered after switching to DR-HC, but there was a significant reduction in the expression and activity of 11β-HSD1 in subcutaneous adipose tissue. 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.

Recycling glucocorticoids: therapeutic implications of the 11β-HSD1 enzyme system.

Endogenous glucocorticoids and commonly used oral glucocorticoids have the property of existing in an inactive and active form in vivo. The inactive form can be converted back to the active form, or 'recycled' in cells and tissues that express the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. This recycling provides an important contribution to the action of glucocorticoids. This review examines the literature relating to the importance of 11β-HSD1 activity during glucocorticoid treatment, with an emphasis on studies examining bone and joint disease and the ability of glucocorticoids to suppress inflammatory damage in models of arthritis. Animal models with global or selective deletion of 11β-HSD1 have determined the extent to which this recycling is important in normal physiology and during treatment with oral glucocorticoids. These studies demonstrate that 11β-HSD1-mediated recycling of inactive glucocorticoids has a substantial action and indeed is responsible for the majority of the effects of orally administered glucocorticoids on a range of tissues. Importantly, the anti-inflammatory actions of glucocorticoids appear largely through this mechanism such that mice that lack 11β-HSD1 are resistant to the anti-inflammatory actions of glucocorticoids. The recognition that to a large extent the circulating inactive counterpart of these glucocorticoids is more important to anti-inflammatory effects than the active glucocorticoid presents novel opportunities to more selectively target glucocorticoids to tissues or to reduce the likely side effects.

3βHSD activity saturates at physiological substrate concentrations in intact cells.

Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) or to Δ5 -androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells. Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. The two reactions displayed very different saturation profiles, with only the 3βHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3βHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD-catalyzed conversion to Δ5 -androstenediol. Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Memory deficits in a juvenile rat model of type 1 diabetes are due to excess 11β-HSD1 activity, which is upregulated by high glucose concentrations rather than insulin deficiency.

Children with diabetes may display cognitive alterations although vascular disorders have not yet appeared. Variations in glucose levels together with relative insulin deficiency in treated type 1 diabetes have been reported to impact brain function indirectly through dysregulation of the hypothalamus-pituitary-adrenal axis. We have recently shown that enhancement of glucocorticoid levels in children with type 1 diabetes is dependent not only on glucocorticoid secretion but also on glucocorticoid tissue concentrations, which is linked to 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity. Hypothalamus-pituitary-adrenal axis dysfunction and memory alteration were further dissected in a juvenile rat model of diabetes showing that excess 11β-HSD1 activity within the hippocampus is associated with hippocampal-dependent memory deficits. Here, to investigate the causal relationships between diabetes, 11β-HSD1 activity and hippocampus-dependent memory deficits, we evaluated the beneficial effect of 11β-HSD1 inhibition on hippocampal-related memory in juvenile diabetic rats. We also examined whether diabetes-associated enhancement of hippocampal 11β-HSD1 activity is due to an increase in brain glucose concentrations and/or a decrease in insulin signalling. Diabetes was induced in juvenile rats by daily i.p. injection of streptozotocin for 2 consecutive days. Inhibition of 11β-HSD1 was obtained by administrating the compound UE2316 twice daily by gavage for 3 weeks, after which hippocampal-dependent object location memory was assessed. Hippocampal 11β-HSD1 activity was estimated by the ratio of corticosterone/dehydrocorticosterone measured by LC/MS. Regulation of 11β-HSD1 activity in response to changes in glucose or insulin levels was determined ex vivo on acute brain hippocampal slices. The insulin regulation of 11β-HSD1 was further examined in vivo using virally mediated knockdown of insulin receptor expression specifically in the hippocampus. Our data show that inhibiting 11β-HSD1 activity prevents hippocampal-related memory deficits in diabetic juvenile rats. A significant increase (53.0±9.9%) in hippocampal 11β-HSD1 activity was found in hippocampal slices incubated in high glucose conditions (13.9 mmol/l) vs normal glucose conditions (2.8 mmol/l) without insulin. However, 11β-HSD1 activity was not affected by variations in insulin concentration either in the hippocampal slices or after a decrease in hippocampal insulin receptor expression. Together, these data demonstrate that an increase in 11β-HSD1 activity contributes to memory deficits observed in juvenile diabetic rats and that an excess of hippocampal 11β-HSD1 activity stems from high glucose levels rather than insulin deficiency. 11β-HSD1 might be a therapeutic target for treating cognitive impairments associated with diabetes.

Dose-Dependent Protective Effect of Hygrophila auriculata Seeds on Cyproterone Acetate-Induced Testicular Dysfunction.

Infertility affects 15% of global population. This study was designed to search out the most effective dose of chloroform fraction of hydro-ethanolic extract of Hygrophila auriculata seed to ameliorate cyproterone acetate (CPA)-treated male subfertility. The rats were made subfertile by CPA at the dose of 2.5 mg/100gm body weight for 45 days. The male subfertility represented by low sperm concentration, less motile, less viable, and less hypo osmotic tail swelled spermatozoa in CPA-treated group. Serum LH, FSH, and testosterone levels were significantly decreased in CPA-treated group in respect to control. Androgenic key enzyme Δ5,3β-HSD, 17β-HSD activities and gene expression pattern were also decreased significantly in respect to control. These antispermatogenic and antiandrogenic activities of CPA were significantly recovered after the treatment of Hygrophila auriculata at the dose of 2.5 mg, 5mg, and 10 mg/100gm body weight. CPA also generate oxidative free radical that indicated by altered catalase, superoxide dismutase, and peroxidase activities and protein expression pattern along with conjugated diene and thiobarbituric acid reactive substance levels in testis. Expression pattern of Bax and Bcl2 genes were deviated from control after CPA treatment. Significant diminution of body weight, organo-somatic indices, and SGOT, SGPT activities were observed in CPA-treated group. All these biomarkers significantly recovered towards control after the treatment of Hygrophila auriculata at different doses. More significant recovery was observed in 5 mg and 10 mg of chloroform fraction-treated group and 5 mg dose, i.e., the minimum therapeutic dose to recover the CPA-induced subfertility.

Abstract #1408256: In Search of New Biomarkers of Thyroid Function: The Association Between Thyroid Dysfunction and 11-beta Hydroxysteroid Dehydrogenase 2 Activity

The high prevalence of persistent hypothyroid symptoms in patients with normal thyrotropin and free thyroxine levels calls for research to identify alternative biomarkers of thyroid function. Animal studies have shown thyroid hormones regulate the renal 11-beta hydroxysteroid dehydrogenase 2 (11βHSD2), a key enzyme in glucocorticoid metabolism. This study examines the impact of hypothyroidism (Overt and subclinical) and hyperthyroidism (overt and subclinical) on 11βHSD2 activity. In this single center prospective study conducted between July 2022-Dec 2022, 24-hour urine samples were collected from 6 patients with newly diagnosed hyperthyroidism, 6 with newly diagnosed hypothyroidism, and 33 age and sex matched healthy reference subjects. A quantitative analysis of 26 steroid metabolites was done in the hydrolyzed urine samples. Given that 11bHSD2 is responsible for the conversion of active cortisol to inactive cortisone, the cortisol to cortisone (F/E) ratio was used to evaluate the enzymatic activity. Differences in total glucocorticoid production and F/E ratios were analyzed based on Z-score deviations from the controls’ reference intervals. The mean participants’ age was 50 years, and 73.3% were female. In patients with hypothyroidism, the mean total glucocorticoid production was 12316.8 ng/mL which was similar to the reference subjects (Z-score -0.03, p = 0.9, CI 95%: -4691.4 to 4666.3), and the mean F/E ratio was 0.5 which was lower than the reference group (Z-score 2.6, p<0.01, CI 95%: 0.06-0.25). In patients with hyperthyroidism, the mean total glucocorticoid production was 16933.7 ng/mL, which was non-statistically higher than the reference subjects (Z-score 1.2, p = 0.1, CI 95%: -794 to 10269), and the mean F/E ratio was 0.4 which was non-statistically higher than the reference group (Z-score 1.6, p = 0.09, CI 95%: 0.01-0.25). After adjustment analysis for total glucocorticoid production, only the differences in the F/E ratio of the hypothyroid patients remained statistically significant (p<0.01). In this proof of principle study, patients with hypothyroidism appeared to have normal total glucocorticoid production but impaired 11βHSD2 activity. Further studies are needed to confirm these findings, determine any clinical implications from cortisol excess in patients with thyroid dysfunction, and evaluate the utility of glucocorticoid metabolomics as a biomarker of peripheral thyroid function action.

Defining corticosteroid-regulated sodium transport within the distal nephron

The aldosterone (ALDO)-sensitive distal nephron is typically defined as: the late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ALDO-sensitivity is defined by 11βHSD2 (11β) activity (which inactivates corticosterone, CORT); mineralocorticoid (MR) rather than glucocorticoid receptor (GR) expression; and the target epithelial sodium channel (ENaC). Recent studies have reported ALDO-insensitive, but MR-dependent ENaC activity in DCT2/CNT and that CORT-stimulated ENaC may be relevant in MR antagonism as 4th line anti-hypertensive therapy. Whilst functional evidence of ALDO-insensitive ENaC activity is compelling, corticosteroid-regulated Na + transport along the distal nephron is not fully defined. AIM: to determine 11βHSD2, MR and GR expression alongside Na + transporter localisation in murine distal nephron and assess the endogenous receptor(s) underpinning steroid-stimulated ENaC activity.Male C57BL/6 mouse kidney sections were subject to multiplex immunofluorescence (11β, MR and GR) and immunohistochemical labelling (NCC, TRPV5, γ-ENaC and AQP2). Images were co-registered, annotated and analysed using Halo® software. Steroid-induced ENaC-mediated currents were measured in mCCD cl1 and primary CD cells. Carbenoxolone (CBX), mifepristone (MF) or PF-03882845 (PF) were used to inhibit 11β, GR and MR, respectively.Distal nephron segments were defined as: DCT - NCC; DCT/CNT - NCC and TRPV5; CNT - TRPV5; CNT/CCD - TRPV5 and γ-ENaC/AQP2; CCD - γ-ENaC/AQP2. We did not detect NCC and γ-ENaC co-localisation. Average cells/section, DCT: 874±39; DCT/CNT: 238±31; CNT: 198±63; CNT/CCD: 831±282; CCD: 2262±231 ( n=3). Negligible numbers of cells expressed both MR and GR.DCT was predominantly 11β- (~90%) and GR dominant (GR:MR ratio ~7:1). In 11β+ cells (~10%), GR was also dominant (GR:MR ratio ~4:1). DCT/CNT and CNT were mainly 11β- (~85 and ~75%, respectively), where MR and GR were similar. In 11β+ cells, MR and GR were similar in DCT/CNT but MR was greater in CNT (MR:GR ratio ~4:1). CNT/CCD contained a near equal split of 11β- (~52%) and 11β+ (~48%) cells. MR and GR were similar in 11β- cells, but 11β+ cells were almost exclusively MR (MR:GR ratio ~37:1). In CCD, ~35% of cells were 11β-, MR and GR were similar, and ~65% cells were 11β+, these were predominantly MR (MR:GR ratio ~30:1).In mCCD cl1 ( n=8-11) and primary CD cells ( n=6), ALDO-induced ENaC activity was inhibited by MR ( p&lt;0.001) but not GR antagonists. DEX-induced ENaC activity was inhibited by GR ( p&lt;0.001) but not MR antagonists. CORT-induced ENaC activity was modestly inhibited by MR ( p&lt;0.05) but not by GR antagonists.Our data suggest that the DCT is a glucocorticoid-responsive segment, with 11β+ cells becoming progressively more dominant from CNT to CCD. The appearance of ENaC in the late CNT and particularly CCD, alongside 11β and MR, suggest these segments reflect the mineralocorticoid-responsive, and thus aldosterone-sensitive distal nephron. Kidney Research UK, The Royal Society This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Functional activity of the adrenal glands in type 2 diabetes patients with different hormonal and metabolic phenotypes

Background. Clinical trials showed a number of additional phenotypes of metabolic syndrome. All of them differ in the type of metabolic disorders and the composition of subcutaneous and visceral adipose tissue. Some of phenotypes have a number of clinical and metabolic similarities with endogenous or exogenous hypercorticism syndromes. The purpose was to characterize the functional activity of the adrenal glands in type 2 diabetes depending on the phenotypic features: the degree of general obesity and the level of visceral fat. Material and methods. Our trial included 89 patients with type 2 diabetes (46 men and 43 women) aged 32 to 85 years. The examination included evaluation of anthropometric parameters, body composition by the bioelectrical impedance method, assessment of the lipid and carbohydrate metabolism, the level of cortisol, dehydroepiandrosterone sulfate (DHEAS) in blood serum, and the activity of 11-beta-hydroxysteroid dehydrogenase (11β-HSD) enzyme. Results. Insulin and C-peptide levels were significantly lower in the non-obese group. The cortisol/DHEAS ratio was elevated in both subgroups with high levels of visceral fat as possible marker of imbalance of anabolic and catabolic hormones. In addition, the concentration of the cortisol, measured in the blood serum of the patients after waking up, was within the normal range. However, the average value in both groups was closer to its upper ranges. This may suggest the presence of subclinical hypercortisolism caused by an increased activity of 11β-HSD, which contributes to the local production of cortisol in visceral adipose tissue. Conclusions. The hormonal and metabolic changes that we found in our groups of patients with type 2 diabetes may indicate anabolic-catabolic imbalance, which is manifested both in the features of the topography of adipose tissue and in changes of metabolic processes, i.e. form the special metabolic phenotype with a catabolic or anabolic axis. Detection the subgroups at high risk allows to develop pathogenetic approaches to the most targeted comprehensive correction of existing violations.

Maternal High-Fructose Corn Syrup Intake Impairs Corticosterone Clearance by Reducing Renal 11β-Hsd2 Activity via miR-27a-Mediated Mechanism in Rat Offspring.

We previously reported that maternal fructose consumption increases blood corticosterone levels in rat offspring. However, the underlying mechanism of action remains unclear. In the present study, we aimed to elucidate the molecular mechanism by which maternal high-fructose corn syrup (HFCS) intake increases circulating GC levels in rat offspring (GC; corticosterone in rodents and cortisol in humans). Female Sprague Dawley rats received HFCS solution during gestation and lactation. The male offspring were fed distilled water from weaning to 60 days of age. We investigated the activities of GC-metabolizing enzymes (11β-Hsd1 and 11β-Hsd2) in various tissues (i.e., liver, kidney, adrenal glands, muscle, and white adipose tissue) and epigenetic modification. 11β-Hsd2 activity decreased in the kidney of the HFCS-fed dams. Moreover, the epigenetic analysis suggested that miR-27a reduced Hsd11b2 mRNA expression in the kidney of offspring. Maternal HFCS-induced elevation of circulating GC levels in offspring may be explained by a decrease in 11β-Hsd2 activity via renal miR-27a expression. The present study may allow us to determine one of the mechanisms of GC elevation in rat offspring that is often observed in the developmental origins of the health and disease (DOHaD) phenomenon.

Curcumin analogues exert potent inhibition on human and rat gonadal 3β-hydroxysteroid dehydrogenases as potential therapeutic agents: structure-activity relationship and in silico docking

Curcuminoids are functional food additives, and the effect on gonadal hormone biosynthesis remains unclear. Gonads contain 3β-hydroxysteroid dehydrogenase isoforms, h3β-HSD2 (humans) and r3β-HSD1 (rats), which catalyse pregnenolone into progesterone. The potency and mechanisms of curcuminoids to inhibit 3β-HSD activity were explored. The inhibitory potency was bisdemethoxycurcumin (IC50, 1.68 µM) >demethoxycurcumin (3.27 µM) > curcumin (13.87 µM) > tetrahydrocurcumin (109.0 µM) > dihydrocurcumin and octahydrocurcumin on KGN cell h3β-HSD2, while that was bisdemethoxycurcumin (1.22 µM) >demethoxycurcumin (2.18 µM) > curcumin (4.12 µM) > tetrahydrocurcumin (102.61 µM) > dihydrocurcumin and octahydrocurcumin on testicular r3β-HSD1. All curcuminoids inhibited progesterone secretion by KGN cells under basal and forskolin-stimulated conditions at >10 µM. Docking analysis showed that curcuminoids bind steroid-active site with mixed or competitive mode. In conclusion, curcuminoids inhibit gonadal 3β-HSD activity and de-methoxylation of curcumin increases inhibitory potency and metabolism of curcumin by saturation of carbon chain losses inhibitory potency.

Synthesis of Novel 2-(Cyclopentylamino)thiazol-4(5H)-one Derivatives with Potential Anticancer, Antioxidant, and 11β-HSD Inhibitory Activities.

In this study, a series of nine new 2-(cyclopentylamino)thiazol-4(5H)-one derivatives were synthesized, and their anticancer, antioxidant, and 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitory activities were tested. Anticancer activity has been assessed using the MTS (MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay against human colon carcinoma (Caco-2), human pancreatic carcinoma (PANC-1), glioma (U-118 MG), human breast carcinoma (MDA-MB-231), and skin melanoma (SK-MEL-30) cancer cell lines. Cell viability reductions, especially in the case of Caco-2, MDA-MB-231, and SK-MEL-30 lines, were observed for most compounds. In addition, the redox status was investigated and oxidative, but nitrosative stress was not noted at a concentration of 500 µM compounds tested. At the same time, a low level of reduced glutathione was observed in all cell lines when treated with compound 3g (5-(4-bromophenyl)-2-(cyclopentylamino)thiazol-4(5H)-one) that most inhibited tumor cell proliferation. However, the most interesting results were obtained in the study of inhibitory activity towards two 11β-HSD isoforms. Many compounds at a concentration of 10 µM showed significant inhibitory activity against 11β-HSD1 (11β-hydroxysteroid dehydrogenase type 1). The compound 3h (2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one) showed the strongest 11β-HSD1 inhibitory effect (IC50 = 0.07 µM) and was more selective than carbenoxolone. Therefore, it was selected as a candidate for further research.

The analysis of pesticides and fungicides in the inhibition of human and rat placental 3β-hydroxysteroid dehydrogenase activity: Mode of inhibition and mechanism

3β-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3β-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3β-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3β-HSD1 and compared them to rat homolog 3β-HSD4. 3β-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3β-HSD1 with half maximal inhibitory concentration (IC50) values of 2.77, 0.24, 0.68, and 17.96 μM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3β-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3β-HSD4 activity with IC50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3β-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3β-HSD4. In conclusion, some pesticides potently inhibit placental 3β-HSD, leading to the reduction of progesterone formation.

Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients

Background and aimConventional drugs have limitations due to prevalence of contraindications in PCOS patients. To explore the potential effects of swertiamarin, on abrupted insulin and steroidogenic signaling in human luteinized granulosa cells from PCOS patients with or without insulin resistance.Experimental procedurehLGCs from 8 controls and 16 PCOS patients were classified for insulin resistance based on down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. Cells were grouped as control, PCOS-IR and PCOS-NIR, treated with swertiamarin (66 µM) and metformin (1 mM). Expression of key molecules involved in insulin signaling, fat metabolism, IGF system and steroidogenesis were compared between groups.ResultsSwertiamarin significantly (P < 0.05) reversed the expression of INSR-β, PI(3)K, p-Akt, PKC-ζ, PPARγ, (P < 0.01) IRS (Ser 307) and IGF system in PCOS-IR group and was equally potent to metformin. In the same group, candidate genes viz SREBP1c, FAS, ACC-1 and CPT-1 were down regulated by swertiamarin (P < 0.001) and metformin (P < 0.001). Significant upregulation was demonstrated in expression of StAR, CYP19A1, 17β-HSD and 3β-HSD when treated with swertiamarin (P < 0.01) and metformin (P < 0.01) in PCOS-IR followed by increase in 17β-HSD and 3β-HSD enzyme activity along with estradiol and progesterone secretions. However, swertiamarin did not reveal any effect on PCOS-NIR group as compared to metformin that significantly (P < 0.01) reversed all the parameters related to steroidogenesis and down regulated basal expression of insulin signaling genes.ConclusionSwertiamarin, presents itself as a potential fertility drug in hLGCs from PCOS-IR patients.

Direct inhibition of human and rat 11β-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 11β-hydroxysteroid dehydrogenase 2 (11β-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 11β-HSD2. C8-C14 PFAS at 100 μM significantly inhibited human 11β-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 11β-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11β-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11β-HSD2 but not rat 11β-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11β-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 11β-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11β-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11β-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11β-HSD2.