11β-HSD1 Activity Research Articles

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.

Direct inhibition of bisphenols on human and rat 11β-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11β-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11β-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 μM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11β-HSD2. Some BPs also inhibited rat renal 11β-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 μM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11β-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11β-HSD2 activity and that there were species-dependent differences.

Health risk assessment, water quality indices and reproductive hormone profile among Abakpa citizens in Enugu metropolis

Numerous substances has been attributed to elicit endocrine-disruption and may potentially affect healthy reproductive functions. This study was carried out to determine the health risk assessment, water quality indices and reproductive hormone profile among citizens of Abakpa in Enugu metropolis. Ten (10) water sampling points comprising of wells and streams were analyzed for chemical properties such BOD, COD, DO, pH, TSS, TDS, Total Hardness, and Alkalinity and Heavy metal composition such as Zinc (Zn), Lead (Pb), Chromium (Cr), Cadmium (Cd) and Mercury (Hg) using the American Public Health Association’s (APHA) guidelines and Atomic absorption spectrophotometer (AAS) respectively. Five (5) blood samples comprising males and females was collected across the sampling areas and assayed for reproductive hormone profile such as Testosterone, Follicle Stimulating Hormone, Progesterone, Prolactin, Estradiol and the enzyme Aromatase was using the ELISA kit method. The result of chemical properties such as TSS, TDS, COD, Total hardness and Alkalinity were all within World Health Organization (WHO) stipulated limits, whereas DO, BOD and pH were higher than WHO limits across all sampling points from the sampling area. Some parameters showed significance difference while some sample did not at different sampling locations at 95% confidence level. The result of heavy metals showed very high concentrations of analyzed metals with Hg present in five water samples from Abakpa at concentrations ranging from 0.01 to 1.96 ppm. Pb concentration ranged between 0.06-0.35 ppm; Zn 24.93- 33.65 ppm; Cr 0.00 – 0.23 ppm; Cd. 0.11 – 0.17 ppm across all sampling points. All the result of reproductive hormone showed lower concentration of testosterone in men (1.99 to 2.19 ng/ml) and high concentration in females (2.03 to 2.06 ng/ml) across all sampling points. The concentration of FSH was in the range of 0.67 to 0.77 ng/ml in males, while the level was between 0.71 to 0.83 ng/ml in female. Prolactin results was 3.49 to 3.86 ng/ml for males and 3.44 to 3.59 ng/ml in females. Estradiol levels were 8.48 to 9.54 ng/ml and 9.61 to 9.84 ng/ml for females and males respectively. However, progesterone level were between 34.08 to 37.45 ng/ml for females and 36.48 to 39.56 ng/ml. The aromatase enzyme was averagely below 4.0 Pmol/mg in both male and female. The risk assessment showed low risk exposure but mercury had high risk exposure tendencies of 16.333 when compared to other heavy metals from all samples. Consumption of water in sampled area’s may pose a serious health challenge; high concentration of heavy metals could be attributed to the hormonal imbalance recorded in the research as most heavy metals is seen to reduce the expression and activity of 3β-HSD and 17β-HSD, which could be facilitated by high acidic nature of the water. Thus, interfering with steroidogenesis. Therefore, continuous monitoring of water quality in Abakpa in Enugu metropolis is highly recommended in order to ensure the health safety of humans living in such environment.

In vitro methods to assess 11β-hydroxysteroid dehydrogenase type 2 activity.

11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts active 11β-hydroxyglucocorticoids to their inactive 11-keto forms, fine-tuning the activation of mineralocorticoid and glucocorticoid receptors. 11β-HSD2 is expressed in mineralocorticoid target tissues such as renal distal tubules and cortical collecting ducts, and distal colon, but also in placenta where it acts as a barrier to reduce the amount of maternal glucocorticoids that reach the fetus. Disruption of 11β-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypernatremia, hypokalemia and hypertension. Secondary hypertension due to 11β-HSD2 inhibition has been observed upon consumption of excessive amounts of licorice and in patients treated with the azole fungicides posaconazole and itraconazole. Furthermore, inhibition of 11β-HSD2 during pregnancy with elevated exposure of the fetus to cortisol can cause neurological complications with a lower intelligence quotient, higher odds of attention deficit and hyperactivity disorder as well as metabolic reprogramming with an increased risk of cardio-metabolic disease in adulthood. This chapter describes in vitro methods for the determination of 11β-HSD2 activity that can be applied to identify inhibitors that may cause secondary hypertension and characterize the enzyme's activity in disease models. The included decision tree and the list of methods with their advantages and disadvantages aim to enable the reader to select and apply an in vitro method suitable for the scientific question and the equipment available in the respective laboratory.

In vitro methods to assess 11β-hydroxysteroid dehydrogenase type 1 activity.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive 11-keto-glucocorticoids to their active 11β-hydroxylated forms. It also catalyzes the oxoreduction of other endogenous and exogenous substrates. The ubiquitously expressed 11β-HSD1 shows high levels in liver and other metabolically active tissues such as brain and adipose tissue. Pharmacological inhibition of 11β-HSD1 was found to ameliorate adverse metabolic effects of elevated glucocorticoids in rodents and humans, improve wound healing and delay skin aging, and enhance memory and cognition in rodent Alzheimer's disease models. Thus, there is an interest to develop 11β-HSD1 inhibitors for therapeutic purposes. This chapter describes in vitro methods to assess 11β-HSD1 enzyme activity for different purposes, be it in disease models, for the assessment of the kinetics of novel substrates or for the screening and characterization of inhibitors. 11β-HSD1 protein expression and preparations of the different biological samples are discussed first, followed by a description of a well-established and easily adaptable 11β-HSD1 enzyme activity assay. Finally, different readout methods are shortly described. This chapter should provide the reader with a toolbox of methods to assess 11β-HSD1 activity with instructions in the form of a decision tree for the choice and implementation of an appropriate enzyme activity assay.

Alteration in glucocorticoids secretion and metabolism in patients affected by cystic fibrosis.

Cystic fibrosis (CF) is an inherited syndrome associated with a mutation in a cystic fibrosis transmembrane conductance regulator gene, composed of exocrine gland dysfunction involving multiple systems that may result in chronic respiratory infections, pancreatic enzyme deficiency, and developmental disorders. Our study describes for the first time the urinary profile of glucocorticoid metabolites and the activity of the enzymes involved in the development and metabolism of cortisol in patients with CF, using a gas chromatography/mass spectrometry method. Data were obtained from 25 affected patients and 70 sex- and age- matched healthy volunteers. We have shown a general decrease in the activity of enzymes involved in the peripheral metabolism of cortisol, such as 11β-hydroxysteroid dehydrogenase type 2, 5α- and 5β-reductases. In contrast, the activity of 11β-hydroxysteroid dehydrogenase type 1, the enzyme that converts cortisone to cortisol, increased. Furthermore, our study found a significant decrease in glucocorticoid excretion in patients with CF. This may suggest adrenal insufficiency or dysregulation of the HPA axis and the development of peripheral mechanisms to counteract cortisol degradation in the case of reduced synthesis of glucocorticoids by the adrenal glands. Furthermore, the activity of 5α-reductase seems to be enhanced only through the backdoor pathway, especially when we taking into consideration 11β-hydroxyandrosterone/11β-hydroxyetiocholanolone ratio which has been shown to be the best differential marker for enzyme activity. CF impairs nutritional effects and energetic balance in patients; thus, our findings suggest the existence of adaptive mechanisms due to limited secretion of adrenal steroids and subsequent diminished amounts of their metabolites in urine. On the other hand, local control of cortisol availability is maintained by enhanced 11βHSD1 activity and its recovery from cortisone in organs and tissues which need this. Steroid hormone dysregulation might be another important factor in the course of CF that should be taken into account when planning an effective and comprehensive therapy.

Inhibition of human and rat placental 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase activities by insecticides and fungicides: Mode action by docking analysis

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 μM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 μM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 μM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 μM significantly inhibited rat HSD3B4 activity, with IC50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 μM, respectively. The mode action analysis showed that triflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference.

Mapping the Transcriptome Underpinning Acute Corticosteroid Action within the Cortical Collecting Duct.

Key Points We report the transcriptomes associated with acute corticosteroid regulation of ENaC activity in polarized mCCDcl1 collecting duct cells.Nine genes were regulated by aldosterone (ALDO), 0 with corticosterone alone, and 151 with corticosterone when 11βHSD2 activity was inhibited.We validated three novel ALDO-induced genes, Rasd1, Sult1d1, and Gm43305, in primary cells isolated from a novel principal cell reporter mouse. Background Corticosteroids regulate distal nephron and collecting duct (CD) Na+ reabsorption, contributing to fluid-volume and blood pressure homeostasis. The transcriptional landscape underpinning the acute stimulation of the epithelial sodium channel (ENaC) by physiological concentrations of corticosteroids remains unclear. Methods Transcriptomic profiles underlying corticosteroid-stimulated ENaC activity in polarized mCCDcl1 cells were generated by coupling electrophysiological measurements of amiloride-sensitive currents with RNAseq. Generation of a principal cell-specific reporter mouse line, mT/mG-Aqp2Cre, enabled isolation of primary CD principal cells by FACS, and ENaC activity was measured in cultured primary cells after acute application of corticosteroids. Expression of target genes was assessed by qRT-PCR in cultured cells or freshly isolated cells after the acute elevation of steroid hormones in mT/mG-Aqp2Cre mice. Results Physiological relevance of the mCCDcl1 model was confirmed with aldosterone (ALDO)-specific stimulation of SGK1 and ENaC activity. Corticosterone (CORT) only modulated these responses at supraphysiological concentrations or when 11βHSD2 was inhibited. When 11βHSD2 protection was intact, CORT caused no significant change in transcripts. We identified a small number of ALDO-induced transcripts associated with stimulated ENaC activity in mCCDcl1 cells and a much larger number with CORT in the absence of 11βHSD2 activity. Principal cells isolated from mT/mG-Aqp2Cre mice were validated and assessment of identified ALDO-induced genes revealed that Sgk1, Zbtbt16, Sult1d1, Rasd1, and Gm43305 are acutely upregulated by corticosteroids both in vitro and in vivo. Conclusions This study reports the transcriptome of mCCDcl1 cells and identifies a small number of ALDO-induced genes associated with acute stimulation of ENaC, including three previously undescribed genes.

ODP321 Impact of a Maternal High-Sucrose Diet in Rats on Fetal Physiology and the Placenta

Abstract Consumption of sucrose (table sugar) is high in much of the world. The effects of maternal sucrose intake on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level alters the mother's physiology and steroids, as well as the adult offspring's brain and behavior. Effects in mothers are impaired glucose tolerance, increased liver lipids, increased adipose inflammation, and decreased corticosterone levels in the blood but not in the brain. In contrast, in adult female offspring, maternal sucrose intake increases corticosterone levels in the blood and the brain. In adult male offspring, maternal sucrose intake increases preference for high-sucrose and high-fat diets as well as motivation for sugar rewards in a progressive ratio task. In this study, we investigate the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. We examine anti-inflammatory steroids, steroidogenic enzymes and cytokines in the placenta, amniotic fluid, fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal serum, placenta, amniotic fluid, fetal blood, and fetal brain. We used Palkovits punch to microdissect the placenta and fetal brain. Next, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is highly sensitive and specific, to measure multiple steroids (e. g. corticosterone, estrone, allopregnanolone). We examined multiple regions of the fetal brain (e. g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). We will also examine neuronal proliferation, microglia and tyrosine hydroxylase immunoreactivity in the fetal brain. Maternal high-sucrose diet increased 11-dehydrocorticosterone (inactive metabolite of corticosterone) and aldosterone in maternal serum and amniotic fluid. Testosterone and androstenedione were significantly higher in the amniotic fluid of male fetuses than female fetuses. Placental steroidogenic enzymes 3β-HSD and CYP19 were not affected by maternal diet or fetal sex; however, 3β-HSD activity was higher in the decidua than in the fetal part of the placenta. Steroid and cytokine data from the placenta, fetal blood and fetal brain are currently being analyzed. Ongoing analyses examine how a maternal high-sucrose diet affects brain development possibly by increasing inflammation. Presentation: No date and time listed

ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells

Abstract Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P&amp;lt;0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis. Presentation: No date and time listed

RF32 | PSUN45 Nicotine E-Cigarette Vapors Exposure Disturbs 11β-Hydroxysteroid Dehydrogenase Type 2 Expression in the Kidney May Contribute to E-Cigarette-Induced Blood Pressure Elevation in Mice

Abstract Inhalation of electronic (e)-cigarettes containing nicotine may elevate blood pressure (BP), but the underlying mechanisms are poorly understood. 11β-HSD2 is crucial for BP regulation by inactivating glucocorticoids in the kidney. Little is known about the effect of e-cigarettes on 11β-HSD2 and their contribution to the process of BP elevation. To examine the possible effects of e-cigarette vapor exposures on renal 11β-HSD2 and BP, four groups of mice were exposed to aerosolized PBS, nicotine-free or nicotine-containing e-cigarettes, with concurrent exposure to either saline or the nicotine antagonist hexamethonium (Hex). Mouse renal M1 CCD cells were used to identify the effects of nicotine on 11β-HSD2 and its transcription factor C/EBPβ. Our results showed that mice exposed to e-cigarettes containing nicotine for 4 weeks markedly decreased the expression and activity of renal 11β-HSD2 and elevated the systolic BP in C57BL/6J mice. Reduction of 11β-HSD2 by nicotine inhalation was accompanied by reduction of renal C/EBPβ and responded to elevated plasma nicotine levels. In contrast, these effects were not observed in mice exposed to e-cigarettes vapor without nicotine compared to PBS controls. Treatment with Hex significantly decreased the elevated ratio of urinary corticosterone to 11-DHC along with stimulation of renal C/EBPβ and prevented the raised BP in mice exposed to e-cigarette vapor containing nicotine. Treating CCD M1 cells with vaporized e-cigarettes containing nicotine extract showed comparable effects with those of pure nicotine for suppression of C/EBPβ and 11β-HSD2, and these effects are blocked by Hex, indicating a mechanistic specificity. Our results indicate that the inhalation of nicotine e-cigarette vapors has potential adverse effects on renal health suggesting that disturbance of renal 11β-HSD2 may contribute to e-cigarette nicotine-induced BP elevation. These findings highlight the potential health risk of e-cigarettes. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 1:00 p.m. - 1:05 p.m.

ODP053 Serum Free Cortisol and Free Cortisol-to-Cortisone Ratio Increase After 10 Days of Overfeeding and After 10 Days of Fasting

Abstract Introduction Chronic caloric deprivation and obesity are complicated by elevations of serum total cortisol levels. The effects of acute overfeeding and fasting on circulating free cortisol levels and interconversion of cortisone to free cortisol are unknown. We hypothesized that serum free cortisol and free cortisol-to-cortisone ratio (a surrogate measure of 11β-hydroxysteroid dehydrogenase [11β-HSD] activity) would increase after both overfeeding and fasting. Methods We prospectively studied 22 healthy volunteers who underwent a 10-day high-calorie protocol followed by a 10-day fast, separated by a 2-week wash-out period, in a Clinical Research Center. Fasting morning free and total cortisol and free cortisone levels (liquid chromatography-tandem mass spectrometry, Mayo Labs) and percent body fat (dual-energy x-ray absorptiometry) were performed at baseline before and after 10 days of each intervention. Results High-calorie feeding increased total and free cortisol and the free cortisol-to-free cortisone ratio (p&amp;lt;0. 001 to p=0. 046). Total and free cortisol, the free cortisol-to-free cortisone ratio, and the free cortisol-to-total cortisol ratio increased after fasting (p=0. 001 to 0. 021). During the high-calorie protocol, there was no significant interaction between sex and time for any of the cortisol parameters. During the fasting visit, the changes in free cortisol and free-to-total cortisol ratio were modified by sex (p=0. 014 and 0. 004, respectively for interaction term), with a trend toward a significant interaction between sex and time in the change in free cortisol-to-free cortisone ratio (p=0. 054). In subset analyses stratified by sex examining the effect of fasting on free cortisol and the free-to-total cortisol ratio, there were significant increases in men (p&amp;lt;0. 001) but not women (p=0.898 and 1. 000, respectively). Baseline percent body fat, measured at the start of the fasting visit, was inversely associated with change in free cortisol (ρ=-0.52, p=0. 013), free cortisol-to-total cortisol ratio (ρ=-0.49, p=0. 021) and free cortisol-to-cortisone ratio (ρ=-0.47, p=0. 029) during fasting. Conclusion Overfeeding and fasting both increase circulating free cortisol levels and appear to alter 11β-HSD activity. The effect of fasting, but not overfeeding, on free cortisol levels is modified by sex. Greater percent fat mass may be relatively protective against starvation-induced hypercortisolemia in women. Further study is warranted to determine whether elevated cortisol levels contribute to complications of starvation and obesity, such as bone fragility. Presentation: No date and time listed

Structure-activity relationship analysis of perfluoroalkyl carbonic acids on human and rat placental 3β-hydroxysteroid dehydrogenase activity

Placenta contains 3β-hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase (HSD3B), which catalyzes pregnenolone to progesterone for maintaining pregnancy. Perfluoroalkyl carbonic acids (PFC) are subclass of perfluoroalkyl substances containing 4–14 carbons (C4-C14) in the carbon backbone and are potential endocrine disruptors. Whether PFC inhibit HSD3B and structure-activity relationship (SAR) remains unclear. Herein, we screened 11 PFC for inhibiting human type I HSD3B (HSD3B1) and rat type IV HSD3B (HSD3B4) activities and determined SAR and mode of inhibition. HSD3B was measured by converting pregnenolone to progesterone assisted by NAD+ in placental microsomes. Of the 11 PFC, C9-C14 significantly inhibited human HSD3B1 activity at 100 μM. Half-maximal inhibitory concentration (IC50) values of C9-C14 compounds were 363.56 ± 12.14, 12.78 ± 0.69, 6.54 ± 0.65, 20.88 ± 0.41, 118.35 ± 0.16, and 149.26 ± 21.67 μM, respectively. We determined Ki values and mode of inhibition of three most potent PFC (C10-C12), and found that they were mixed inhibitors against pregnenolone, with Ki values of 5.57 ± 4.37, 2.04 ± 2.26, and 9.93 ± 7.71, respectively. Docking analysis showed that they bound steroid-binding site. Effects of PFC on rat placental HSD3B4 were performed. Of the 11 PFC, C10-C12 significantly inhibited rat HSD3B4 activity at 100 μM. IC50 values of C10-C12 compounds were 45.85 ± 1.49, 36.08 ± 1.50, and 88.74 ± 1.99 µM, respectively. Ki values and inhibition modes of the three most potent PFC (C10-C12) were studied. It was found that they were mixed inhibitors against pregnenolone, with Ki values of 48.16 ± 20.44, 36.28 ± 53.07, and 91.79 ± 21.75 μM, respectively. Docking analysis showed that they bound steroid-binding site of rat HSD3B4. In conclusion, PFC showed significant SAR differences. The potency of inhibiting HSD3B activity increased from C9 to C11, and then declined. Human HSD3B1 was more sensitive to the inhibition of rat HSD3B4.

Sex-specific endocrine regulation of seasonal aggression in Siberian hamsters.

Coordinating physiological and behavioural processes across the annual cycle is essential in enabling individuals to maximize fitness. While the mechanisms underlying seasonal reproduction and its associated behaviours are well characterized, fewer studies have examined the hormonal basis of non-reproductive social behaviours (e.g. aggression) on a seasonal time scale. Our previous work suggests that the pineal hormone melatonin facilitates a 'seasonal switch' in neuroendocrine regulation of aggression in male and female Siberian hamsters (Phodopus sungorus), specifically by acting on the adrenal glands to increase the production of the androgen dehydroepiandrosterone (DHEA) during the short-day (SD) photoperiods of the non-breeding season. Here, we provide evidence that the activity of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), a key enzyme within the steroidogenic pathway that mediates DHEA synthesis and metabolism, varies in a sex-specific and melatonin-dependent manner. Although both male and female hamsters displayed increased aggression in response to SDs and SD-like melatonin, only males showed an increase in adrenal 3β-HSD activity. Conversely, SD and melatonin-treated females exhibited reductions in both adrenal and neural 3β-HSD activity. Collectively, these results suggest a potential role for 3β-HSD in modulating non-breeding aggression and, more broadly, demonstrate how distinct neuroendocrine mechanisms may underlie the same behavioural phenotype in males and females.

Curcumin Remedies Testicular Function and Spermatogenesis in Male Mice with Low-Carbohydrate-Diet-Induced Metabolic Dysfunction.

Increasing reports on the significance of dietary patterns in reproduction have arisen from both animal and human studies, suggesting an interactive association between nutrition and male fertility. The aim of this study was to investigate the effects of curcumin supplementation on low-carbohydrate-diet-induced metabolic dysfunction, testicular antioxidant capacity, apoptosis, inflammation and spermatogenesis in male mice. Male C57BL/6 mice were fed a normal diet (AIN-93M group, n = 12) and a low-carbohydrate diet for 12 weeks (LC group, fed with low-carbohydrate diet, n = 48), and mice randomly chosen from the LC group were later fed their original diet (LC group, n = 12). This diet was changed to AIN-93M feed (LC/AIN-93M group, n = 12), a ketogenic diet (LC/KD group, n = 12), or a ketogenic diet treated with curcumin supplementation for the final 6 weeks (LC/KDCu group, n = 12). A poor sperm morphology and mean testicular biopsy score (MTBS) were observed in the LC and LC/KD groups, but they were eliminated by the normal diet or ketogenic diet with curcumin. The LC group exhibited a lower testicular testosterone level and a lower 17β-HSD activity and protein expression. This also enhanced apoptosis protein expressions in testis tissue, including Bax/BCl2, cleaved caspase 3, PARP and NF-κB. Meanwhile, we found a statistically significant increase in lipid peroxidation and decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase levels in the LC group. Our study indicated that a replacement of a normal diet or ketogenic diet supplemented with curcumin attenuated poor semen quality and reduced testosterone levels by the LC diet by reducing oxidative stress.

Metformin alleviates the dysregulated testicular steroidogenesis and spermatogenesis induced by carbimazole in levothyroxine-primed rats

Most of the published experiments about carbimazole (CMZ)-induced testicular injury are constructed in normal healthy animals, which lakes the translational identification. Despite metformin (MET) having advantageous effects on injured testicles, its impact on thyroid function is arguable. In the current levothyroxine (LT4)/CMZ model, Wistar rats were primed by LT4 for sixty days. CMZ was then given individually or simultaneously with different doses of MET, 100, 200, and 400 mg, daily for thirty days. Serum was assessed for thyroid profile panel, sex hormones, and gonadotropin levels. Testicular tissues were examined for steroidogenesis, spermatogenesis, inflammation, and apoptosis. Histopathology of thyroid and testes were examined, besides thyroidal nuclear factor (NF)-kB expression. MET in a dose-response manner improved the LT4/CMZ-induced testicular toxicity by increasing the steroidogenic acute regulatory protein (StAR), and 17-β-hydroxysteroid dehydrogenase (17βHSD) activities, the proliferating cell nuclear antigen (PCNA), sperm count and motility, sex hormones, and gonadotropin levels. MET-400 mg markedly decreased the elevated NF-kB expressions, tumour necrosis factor (TNF)-α, caspase-3, and BAX, and increased BCL-2. LT4/CMZ could be used as translational animal modelling. MET displayed a dose-dependent ameliorative effect on the LT4/CMZ model without significant harmful effects on thyroid functions. MET-testicular protective roles in diabetics with thyroidal diseases should be explored.

Increased systemic and adipose 11β-HSD1 activity in idiopathic intracranial hypertension.

ContextIdiopathic intracranial hypertension (IIH) is a disease of raised intracranial pressure (ICP) of unknown etiology. Reductions in glucocorticoid metabolism are associated with improvements in IIH disease activity. The basal IIH glucocorticoid metabolism is yet to be assessed.ObjectiveThe objective of this study was to determine the basal glucocorticoid phenotype in IIH and assess the effects of weight loss on the IIH glucocorticoid phenotype.DesignA retrospective case–control study and a separate exploratory analysis of a prospective randomized intervention study were carried out.MethodsThe case–control study compared female IIH patients to BMI, age, and sex-matched controls. In the randomized intervention study, different IIH patients were randomized to either a community weight management intervention or bariatric surgery, with patients assessed at baseline and 12 months. Glucocorticoid levels were determined utilizing 24-h urinary steroid profiles alongside the measurement of adipose tissue 11β-HSD1 activity.ResultsCompared to control subjects, patients with active IIH had increased systemic 11β-hydroxysteroid dehydrogenase (11β-HSD1) and 5α-reductase activity. The intervention study demonstrated that weight loss following bariatric surgery reduced systemic 11β-HSD1 and 5α-reductase activity. Reductions in these were associated with reduced ICP. Subcutaneous adipose tissue explants demonstrated elevated 11β-HSD1 activity compared to samples from matched controls.ConclusionThe study demonstrates that in IIH, there is a phenotype of elevated systemic and adipose 11β-HSD1 activity in excess to that mediated by obesity. Bariatric surgery to induce weight loss was associated with reductions in 11β-HSD1 activity and decreased ICP. These data reflect new insights into the IIH phenotype and further point toward metabolic dysregulation as a feature of IIH.

11β-Hydroxysteroid Dehydrogenase Type 1 within Osteoclasts Mediates the Bone Protective Properties of Therapeutic Corticosteroids in Chronic Inflammation.

Therapeutic glucocorticoids (GCs) are powerful anti-inflammatory tools in the management of chronic inflammatory diseases such as rheumatoid arthritis (RA). However, their actions on bone in this context are complex. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a mediator of the anti-inflammatory actions of therapeutic glucocorticoids (GCs) in vivo. In this study we delineate the role of 11β-HSD1 in the effects of GC on bone during inflammatory polyarthritis. Its function was assessed in bone biopsies from patients with RA and osteoarthritis, and in primary osteoblasts and osteoclasts. Bone metabolism was assessed in the TNF-tg model of polyarthritis treated with oral GC (corticosterone), in animals with global (TNF-tg11βKO), mesenchymal (including osteoblast) (TNF-tg11βflx/tw2cre) and myeloid (including osteoclast) (TNF-tg11βflx/LysMcre) deletion. Bone parameters were assessed by micro-CT, static histomorphometry and serum metabolism markers. We observed a marked increase in 11β-HSD1 activity in bone in RA relative to osteoarthritis bone, whilst the pro-inflammatory cytokine TNFα upregulated 11β-HSD1 within osteoblasts and osteoclasts. In osteoclasts, 11β-HSD1 mediated the suppression of bone resorption by GCs. Whilst corticosterone prevented the inflammatory loss of trabecular bone in TNF-tg animals, counterparts with global deletion of 11β-HSD1 were resistant to these protective actions, characterised by increased osteoclastic bone resorption. Targeted deletion of 11β-HSD1 within osteoclasts and myeloid derived cells partially reproduced the GC resistant phenotype. These data reveal the critical role of 11β-HSD1 within bone and osteoclasts in mediating the suppression of inflammatory bone loss in response to therapeutic GCs in chronic inflammatory disease.

Stress and adaptation to repeated stress changed the expression of two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) in the adrenal glands of sheep

The aim of this study was to estimate rate of cortisol release from ewe’s adrenal gland cortex and medulla tissue and the participation 11β-HSD1 and 2 in modulating adrenal glucocorticosteroids release, determine the expression size of these two isoforms in both parts of adrenal glands after single and repeated stress (3 times every 24h), when adaptive processes take place. The experiment was carried on 15 ewes, outside the breeding season. The research material consisted of adrenal glands obtained from control sheep (CON), sheep subjected to one-hour individual isolation from the herd (S1) and three times repeated stress factor at one-day intervals (S2). Immediately after slaughter, the adrenal glands from each sheep were placed on ice in 0.9% NaCl, then they were cut into smaller sections of similar weight (approx. 50 mg), covering the cortical and medullary layers of the adrenal glands. Sections of obtained tissues were weighed and placed in incubation wells (Sigma cell culture) containing 1 ml of incubation Eagle's medium with the addition of bovine albumin (BSA, 50 mg / 100 ml). Tissue incubation was carried out in an atmosphere of carbogen - 95% O2 and 5% CO2 at 38oC in a Sanyo incubator (MCO-18AIC). After 10 minutes of stabilization, the sections of core and adrenal cortex tissue were transferred to subsequent wells containing pure Eagle Medium. The same section was transferred to consecutive incubation wells containing clean incubation medium every 30 minutes. The medium harvested from the wells after 30, 60 and 90 minutes of incubation of adrenal tissue was frozen until the scheduled cortisol concentration de-termination by RIA (Orion Diagnostica) was performed. Another pieces of tissue the cortex and adrenal medulla from each sheep immediately placed in the RNAlater. Measurements of the gene expression of 11β-HSD1 and 2 expression in the adrenal cortex and medulla were performed using Real-time qPCR method. Starters and TaqMan MGB probes were designed by Applied Biosystem. In the conducted in vitro experiment, a significant reduction in the amount of cortisol released from adrenal cortex tissue into the medium after single stress factor was found, from 3.27 ± 0.69 pg･mg-1 tissue after first 30 minutes of experiment to 0.70 ± 0.15 pg･mg-1 of tissue after 90 minutes (P &lt; 0.01). A similar profile of cortisol release from the adrenal cortex tissue was found in control sheep (P &gt; 0.05). Experimental Group 2 (S2) had a similar profile of cortisol release during the all-time of incubation, however the values found were always lower than those observed in the control and single stress sheep Experimental Group 1 (S1) (1.34 ± 0.37 to 0.30 ± 0.08 pg･mg-1 of tissue; P &lt; 0.05). After repeated stress, incubation of adrenal cortex tissue showed a similar profile of changes in cortisol release, however the values were significantly lower than those found in the singly stressed animals (from 1.30 ± 0.37 pg･mg-1 after 30 minutes to 0.27 ± 0.08 pg･mg-1 tissue after 90 minutes; P &lt; 0.01). Incubation of adrenal medulla tissue obtained from singly stressed animals showed cortisol release to the medium much lower in comparison to adrenal cortex layer, however constant during the whole incubation time, value 1.04 ± 0.36 pg/mg-1 tissue noted after first 30 minutes to 0.77 ± 0.06 pg･mg-1 after 90 minutes of experiment; P &gt; 0.05). When the stress factor was repeated three times, the amount of cortisol released was like that observed in the control secretion; P &gt; 0.05). Analysis of 11β-HSD 1 expression in adrenal cortex tissue from singly stressed sheep showed an over threefold increase (350 ± 25 RQ), while measurement after stress factor was repeated three times demonstrated increase, however the value turned out to be two times lower than after a single stress (160 ± 35 RQ). The expression value of 11β-HSD 2 in the adrenal cortex tissue showed a significant reduction after both single (15 ± 7 RQ and repeated stress (22 ± 3.0 RQ). No significant differences were found in the expression value of this enzyme in the sheep adrenal medulla in both experimental groups in comparison to control group (P &gt; 0.05). The obtained results indicate the immanent abilities of the adrenal glands, probably to a certain extent, to control the release of cortisol, conditioned by the presence and variable activity of the enzyme 11β-HSD, particularly isoform 1, in adrenal glands without the need to activate all HPA axis structures during stress and adaptation processes.