ResearchPad - steroid-biology-and-action https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[OR09-03 Brain Aromatase Is Essential for Regulation of Sexual Activity in Male Mice]]> https://www.researchpad.co/article/elastic_article_8737 Introduction: The biologically active form of estrogen, estradiol (E2), has important organizational roles in brain development and activational roles in adult brain physiology and behavior. It has been proposed that E2 formation in the brain might regulate sexual activity in various species. The mechanisms that link estrogen formation in the brain and sexual behavior, however, remain unclear. Aromatase is the key enzyme that catalyzes the conversion of testosterone (T) to E2 in the testis and brain of male mice. To determine the role of brain aromatase in male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse model. Additionally, a newly generated total aromatase knockout (tArKO) mouse model served as a positive control. Methods: We generated the floxed aromatase mice (Aromfl/fl), which flanked the transcription and translation start sites and the common splice acceptor site for the upstream brain promoter I.f of the aromatase gene. We then crossed Nestin-Cre mice with Aromfl/fl mice to generate bArKO mice. Using the same Aromfl/fl mice, we bred tArKO via crossing with ZP3-Cre mice. Circulating and tissue (brain and testis) E2 levels were measured using liquid chromatography-tandem mass spectrometry. We assessed sexual activity in 12-14 week-old bArKO, tArKO and littermate control males over two 30-minute trials. The interactions were monitored and videotaped, and the videotape was scored for the sexual activity. To investigate whether the lack of estrogen production in the brain was causative for altered sexual behavior, 20 bArKO and 20 control mice were castrated at ~nine weeks of age and supplemented with exogenous sex hormone via 60-day time release pellet implantation. Results: E2 levels are significantly decreased in the brain but not the testis of bArKO mice as compared to control mice (P < 0.05, n=6-12). As expected, E2 levels in the brain and testis are significantly lower in tArKO mice compared with their WT littermates (n=6-9). Furthermore, we demonstrate that local aromatase expression and estrogen production in the brain is required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited significantly decreased sexual activity in the presence of strikingly elevated circulating T (n=5). In castrated adult bArKO mice, administration of E2 together with T restored maximum sexual behavior (n=5). Thus, aromatase in the brain is necessary for T-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating T of testicular origin. Conclusion: Our findings suggest T activates male sexual behavior in part via conversion to E2 in the brain and provide the foundation for inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators in modifying sexual behavior.

DCB and HZ contributed equally to this work.

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<![CDATA[SAT-737 Low-Dose Testosterone Augmentation for Treatment-Resistant Depression in Women: An 8-Week, Two-Site, Randomized, Placebo-Controlled Study]]> https://www.researchpad.co/article/elastic_article_7156 Objective: Nonresponse to selective serotonin reuptake inhibitor and serotonin norepinephrine reuptake inhibitor treatment is common in patients with major depressive disorder (MDD), particularly in women, occurring in about 70% of patients despite adequate dosing. Well-tolerated augmentation strategies are needed, particularly ones that do not cause or exacerbate symptoms such as fatigue and sexual dysfunction. Low-dose testosterone has been shown to improve depression symptom severity, fatigue and sexual function in small studies of women not formally diagnosed with MDD. We sought to determine whether adjunctive low-dose transdermal testosterone improves depression symptom severity, fatigue, and sexual function in women with treatment-resistant MDD. A functional MRI (fMRI) substudy examined effects of testosterone on activity in the anterior cingulate cortex (ACC), a brain region important in mood regulation.

Methods: Randomized, double-blind, placebo-controlled, 8-week trial of adjunctive testosterone cream (AndroFeme® 1, Lawley Pharmaceuticals, Australia) in 101 women, ages 21–70, with treatment-resistant MDD. Testosterone was titrated to achieve blood levels near the upper normal reference limit. Primary outcome measure was depression severity by Montgomery-Asberg Depression Rating Scale (MADRS). Secondary endpoints included fatigue, sexual function, and safety measures. fMRI substudy (n=20) primary outcome was change in ACC activity.

Results: Mean age was 47±14 (SD) years and mean baseline MADRS score was 26.6±5.9. Eighty-seven (86%) participants completed 8 weeks of treatment. MADRS depression scores decreased in both arms [testosterone: 26.8±6.3 to 15.3±9.6; placebo: 26.3±5.4 to 14.4±9.3 (baseline to 8 weeks, respectively)], with no difference between groups (p=0.91). Fatigue and sexual function improved without differences between groups. There were no group differences in side effects. fMRI results demonstrated a relationship between ACC activation and androgen levels pretreatment but no difference in ACC activation with treatment.

Conclusions: This rigorously designed, double-blinded clinical trial did not find significant group differences between adjunctive low dose transdermal testosterone and placebo for antidepressant augmentation in women with treatment-resistant MDD and had a high placebo response rate. Low-dose testosterone was well tolerated but failed to differentially impact overall depressive symptom severity, fatigue, or sexual dysfunction. Testosterone did not result in greater activity in a brain region (ACC) implicated in MDD etiopathology compared to placebo. Thus, the addition of low-dose testosterone to ineffective antidepressant treatment should not be recommended for women with MDD. Further studies using strategies designed to reduce placebo effects may be warranted.

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<![CDATA[SAT-736 Dissecting the Relative Role of Estrogen and Androgen in Fibrosis, Skeletal Muscle Atrophy, and Inguinal Hernia Formation]]> https://www.researchpad.co/article/elastic_article_6965 Introduction: More than one in four men develop symptomatic inguinal hernia, and hernia repair is the most commonly performed general surgical procedure in the US. Despite its prevalence, the molecular mechanisms causing inguinal hernia remain unclear. Aromatase, the key enzyme for the conversion of testosterone (T) to estradiol (E2), is present in human but not mouse skeletal muscle tissue. We recently demonstrated that robustly increased local E2 levels in lower abdominal muscle (LAM) tissue and decreased circulating T levels were associated with fibrosis and myocyte atrophy in LAM tissue, leading to severe scrotal (inguinal) hernia formation in a humanized aromatase transgenic mouse model (Aromhum) with a high LAM human aromatase expression. To further determine the relative role of estrogen and androgen in the development of inguinal hernia, we generated a novel mild Aromhum mouse model with lower LAM aromatase expression compared with the severe model. Methods: Mild Aromhum mice were followed for 6 months to determine hernia incidence and measure hernia size (n=30). We treated mild Aromhum mice with the aromatase inhibitor, letrozole (n=12) for 12 weeks. Circulating and LAM E2 levels in mice were measured using mass spectrometry. LAM tissue fibrosis and myocyte size were determined by Masson’s trichrome staining and H&E staining, respectively. Results: The mild Aromhum mice contain a single copy of the human aromatase genomic fragment with a truncated regulatory region, giving rise to significant but mildly elevated LAM E2 levels (2.5-fold) at 15 weeks of age. Interestingly, these mice maintain normal circulating T levels. Furthermore, we show that mildly increased LAM E2 without decreased circulating T levels cause hernia formation in about 88% of mild Aromhum mice in contrast to 100% hernia formation in mice containing the full-length human aromatase regulatory region (severe Aromhum model), suggesting that higher LAM estrogen and low serum T levels contribute to this severe phenotype. Treatment with an aromatase inhibitor restores LAM E2 levels to normal levels and completely prevents inguinal hernia formation in the mild Aromhum mice. In LAM fibroblasts of mild Aromhum mice, we find very high levels of estrogen receptor-α expression, which possibly mediates estrogen-induced hernia formation. Conclusion: Taken together, our findings from the mild Aromhum mouse model suggest that lower levels of estrogen excess in LAM are the primary driver of muscle atrophy and hernia formation because this mouse model do not exhibit circulating T deficiency. Our findings will constitute a starting point for dissecting the relative roles of estrogen and androgen action in inguinal hernia development. This has the potential to facilitate drug development to prevent and treat hernias, especially recurrent hernias after primary hernia repairs in vulnerable populations such as elderly men.

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<![CDATA[SAT-LB135 A Novel Cytoplasmic Membrane Estrogen-Mediated Biogenic Signaling Pathway]]> https://www.researchpad.co/article/elastic_article_6664 The estrogen receptor α (ERα) is known to convey both genomic and extra-genomic activities. The extra-nuclear estrogen signaling pathway is thought to involve a membrane-associated estrogen receptor (ERα), which activates PI3-kinase and Akt signaling. Maximal activation of Akt requires S473 phosphorylation. The essential G1-cyclin, CCND1, is a collaborative nuclear oncogene that is frequently overexpressed in cancer. D-type cyclins bind and activate CDK4/6, contributing to G1-S cell-cycle progression. Herein, cyclin D1 was shown to be located in the cytoplasmic membrane of patients with inflammatory breast cancer, human diploid fibroblasts and cancer cell lines (breast, prostate). The extra-nuclear vs. nuclear E2-induced signaling pathways can be distinguished using 17β-estradiol linked to a dendrimer conjugate (EDC), which excludes estradiol from the nucleus. In contrast with the nuclear-localized form of cyclin D1 (cyclin D1NL), the cytoplasmic membrane-localized form of cyclin D1 (cyclin D1CML) was sufficient to induce phosphorylation of the serine threonine kinase Akt (Ser473) and augmented extra-nuclear localized 17β-estradiol dendrimer conjugate (EDC)-mediated phosphorylation of Akt (Ser473). Cyclin D1CML was sufficient to induce G1-S cell-cycle progression, cellular proliferation, colony formation. In contrast with cyclin D1NL, the cyclin D1CML induced transwell migration and the velocity of cellular migration. Together these studies suggest distinct subcellular compartments of cell cycle proteins may convey distinct functions. The major adjuvant therapy for the ~70% of ERα expressing human breast cancer involves anti-estrogen therapy and the ERα/PI3K/Akt complex pathway is hyperactivated in aggressive breast tumors. The non-genomic actions of E2/ERα, mediated via cyclin D1CML may provide an important additional target. References. 1. 2. Casimiro MC et al Mol Endocrinol. 2013;27(9):1415-28. Di Sante, G, Expert Rev Anticancer Ther. 2019 Jun 20:1-19.

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<![CDATA[SAT-749 Defining The Role Of Androgens In Hernia Associated Skeletal Muscle Fibrosis]]> https://www.researchpad.co/article/elastic_article_6534 Introduction: Inguinal hernia is a highly prevalent condition occurring in 27% of adult men in their lifetime. The recurrence rate of hernia is 5-20%, resulting in a substantial cost burden in surgical repair procedures. Until recently, the mechanisms leading to the lower abdominal muscle (LAM) weakening characteristic of hernia were unknown. Our group developed the first mouse model of inguinal hernia through expression of the human aromatase enzyme in male mice (AromHum). Aromatase converts androgens to estrogens, and is expressed in the skeletal muscle in humans, but not mice. We found that locally formed estrogen from aromatase activity in LAM and decreased circulating testosterone levels are associated with muscle atrophy and fibrosis resulting in hernia. However, it is unclear how decreasing androgen levels might affect muscle fibrosis, and defining this potential mechanism could impact hernia treatment. We hypothesized that low androgen levels promote muscle fibroblast proliferation and fibrosis, and that androgen treatment would prevent hernia progression in AromHum mice.

Methods: Arom Hum mice (3 weeks old) were treated with high-dose dihydrotestosterone (DHT) via injection for 7.5 weeks with hernia volume continuously recorded (n=5/group). Primary fibroblasts were isolated from LAM from WT and AromHum mice (n=5/genotype). Cells were treated for 24 hours with increasing doses (0.001, 0.01, 0.1, 1, 5, 10 and 100 nM) of R1881, a synthetic androgen, and compared to untreated cells by western blot.

Results: Hernia volume was significantly decreased in AromHum mice treated with DHT compared to vehicle-treated mice, and volume remained consistently suppressed after DHT treatment (p < 0.005). In both primary fibroblast lines, R1881 treatment increased AR levels in a dose dependent manner, indicating that the treatment was effective. Preliminary data indicated that low doses of R1881 (0.001 and 0.01 nM) increased PCNA levels in LAM WT and LAM AromHum fibroblasts. Densitometry normalized to GAPDH showed 80% and 60% increases for 0.001 nM and 0.01 nM respectively in LAM WT fibroblasts, and 20% and 30% increases at these doses in LAM AromHum fibroblasts. Higher doses of R1881 decreased PCNA levels in LAM AromHum fibroblasts by 40% (10 nM) and 30% (100 nM), whereas a 25% decrease was detected in LAM WT fibroblasts at 100 nM.

Conclusion: These data suggest that low androgen doses increase LAM fibroblast proliferation, which possibly contributes to hernia formation. Androgen treatment at higher doses can partially block the progression of hernia in vivo. However, it is unclear whether and how androgen deficiency in combination with excess estrogen affects fibroblast proliferation and hernia formation. Additional research is required to determine if androgen supplementation in sufficient doses is a potential therapeutic for inguinal hernia and other muscle weakness diseases.

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<![CDATA[SAT-733 Improving the Diagnosis, Treatment, and Prevention of Endocrine Diseases Through Accurate and Reliable Laboratory Measurements with CDC’s Clinical Standardization Programs]]> https://www.researchpad.co/article/elastic_article_6419 Laboratory measurements are critical for the correct diagnosis and treatment of patients as well as in the investigation of chronic diseases such as hypogonadism, PCOS, and bone-and kidney-related diseases. Inaccurate measurements can lead to misclassification of patients and incorrect treatment. Furthermore, the effective use of research findings in patient care is prevented. The CDC Clinical Standardization Programs (CDC CSP) assess the analytical performance of assays against performance goals defined by clinical and medical organizations. The CDC CSP assist with assay calibration, the certification of analytical performance, and the monitoring of analytical performance during the measurement of patient and/or study samples. CDC CSP have programs in place for the calibration and certification of commercial assays and laboratory developed tests (LDTs) for total testosterone (TT), estradiol (E2), vitamin D (VD), free thyroxine (FT4), total cholesterol (TC), total glycerides (TG), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C). The programs available for monitoring analytical performance during routine testing include TT, VD, TC, TG, HDL-C, apolipoprotein AI and B. CDC CSP also support accuracy-based external quality assurance surveys such as those offered by the College of American Pathologists. Enrollment of assays and LDTs in CDC’s certification programs has resulted in improvements in calibration accuracy; i.e. the absolute mean bias of assays participating in the CDC Vitamin D Standardization Certification Program was well below the allowable bias of 5% each year. Assays standardized in CDC’s certification programs also demonstrated higher accuracy in routine patient testing; i.e. CDC VD certified assays have a lower bias compared to non-certified assays. Similar observations were made with assays certified in the CDC’s program for TT. Monitoring data over the past 10 years from the CDC Lipid Standardization Program indicated that the majority of TC measurements performed in routine testing were consistently within the recommended bias limits of ±3%. CDC CSP continue to improve the analytical performance of assays by addressing measurement bias caused by factors other than incorrect calibration such as interfering compounds. The programs are responding to new clinical and public health needs with the addition of new analytes such as PTH and glucose. The CDC CSP support projects aiming at establishing reference intervals and other research studies. The CDC CSP work with stakeholders, such as the Partnership for the Accurate Testing of Hormones and the Endocrine Society, to educate the clinical and laboratory communities about the importance of using standardized assays in patient care, research, and public health. References: Partnership for Accurate Hormone Testing (PATH). www.hormoneassays.org. College of American Pathologists (CAP). www.cap.org.

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<![CDATA[OR09-06 HNRNPA2B1 Mediates Endocrine-Sensitivity and Alters PSAT1 in Breast Cancer Cells]]> https://www.researchpad.co/article/elastic_article_6162 Higher expression of the RNA binding protein HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), a reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is found in endocrine-resistant, estrogen receptor (ERα)+ LCC9 and LY2 breast cancer cells derived from MCF-7 endocrine-sensitive luminal A cells (1). HNRNPA2B1 interacts with DGCR8 in the DROSHA complex to promote processing of pri-miRNAs to pre-miRNAs. We identified HNRNPA2B1-regulated miRNAs by RNA seq and target pathways, including serine family amino acid metabolic processes, TGFβ signaling, response to estrogen, and cell junction by MetaCore enrichment pathway analysis (1). Stable 4.5-fold overexpression of HNRNPA2B1 in MCF-7 cells (MCF-7-A2B1 cells) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased ~ 1.6-fold. Conversely, transient knockdown of HNRNPA2B1 expression in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant, without changing ESR1 expression. MCF-7-A2B1 cells showed increased migration, reduced E-cadherin and increased vimentin, suggestive of EMT; however, they exhibited reduced clonogenic survival. Follow-up on the identification of HNRNPA2B1-miRNA regulation of the serine pathway revealed higher expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) transcripts and proteins in LCC9, LY2, and MCF-7-A2B1 compared to MCF-7 cells. We identified two miRNAs, miR-424-5p and miR-145-5p downregulated in MCF-7-A2B1 cells that directly targeted the PSAT1 3’UTR in dual luciferase assays. Lower miR-424-5p and miR-145-5p in endocrine-resistant LCC9 and LY2 correlate with increased PSAT1 and higher PSAT1 is associated with reduced overall and metastasis-free survival in breast cancer patients. Overall, our data suggest a role for increased HNRNPA2B1 in tamoxifen-resistance. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Sci. Rep. 2019; 9:9430.

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<![CDATA[SAT-741 Salivary Cortisol and Cortisone Measurement Provide a Novel and Non-Invasive Method of Monitoring Medical Therapy in Cushing’s Syndrome]]> https://www.researchpad.co/article/elastic_article_6122 Introduction:

Salivary glucocorticoids (cortisol [SalF], cortisone [SalE]) are collected non-invasively, unaffected by variation in cortisol binding globulin (CBG) and an established tool in the investigation of Cushing’s syndrome (CS). We have previously shown a strong correlation between salivary glucocorticoids and circulating free serum cortisol, better than that with total serum cortisol. Measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS) permits lower limits of quantification, eliminates cross-reactivity both between cortisol and cortisone, and by metyrapone-induced elevations in 11-deoxycortisol.

Previous studies in CS have demonstrated that a mean (based on 5 samples during a single day) serum cortisol (serumF) of 150-300 nmol/l equates to a normal isotopically calculated cortisol production rate. We report the use of salivary glucocorticoid measurement in metyrapone-treated CS patients undergoing dose titration to achieve a mean serumF of 150-300 nmol/L.

Methods:

Seventeen (11 females; age-range 24-74 years) patients with CS undergoing dose titration with metyrapone were studied on 44 occasions: 15 ACTH-dependent (5 ectopic) and 2 adrenal.

24 healthy male volunteers (HV) were also studied.

Both cohorts had paired serumF and SalE and SalF samples collected at 5 time-points (10:00; 11:30; 13:00; 14:30; 16:00).

Serum and salivary glucocorticoids were measured using LC-MS/MS.

The TR for salivary glucocorticoids was determined from the mean SalE and SalF in HV whose mean serumF (n=20) was in the desired range (150-300 nmol/L). In CS patients the metyrapone dose had been titrated to achieve a mean serumF of 150-300 nmol/L on 14 (out of 44) occasions.

Results:

Mean SalE (r=0.70; p<0.001) and mean SalF (r=0.51; p<0.001) showed a significant correlation with serumF. The TR from HV for mean SalE and mean SalF were 5.8–24.7 nmol/L and 0.6-5.4 nmol/L respectively.

In CS patients, SalE had greater sensitivity (79% vs. 75%) and specificity (80% vs. 57%) in predicting a mean serumF in the TR than SalF.

Conclusion:

We have demonstrated a close correlation between mean SalE and mean serumF in metyrapone treated CS patients. Salivary glucocorticoids within the derived TR were highly predictive for a target serumF. It is unsurprising, due to the effect of CBG on serumF measurements, that the sensitivity and specificity of SalE and SalF are not greater than reported. Consequently, SalE and SalF, as surrogates for free serum F, have the potential to be superior than serumF when assessing adequacy of medical therapy in CS and may permit out-of-hospital monitoring. Further work is required to validate these findings.

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<![CDATA[SAT-747 A Prospective Non-surgical Treatment for Inguinal Hernias]]> https://www.researchpad.co/article/N535c7efa-9da7-4dfd-9c09-99260a4707ae <![CDATA[SAT-739 Membrane Progesterone Receptors (mPRs/PAQRs) Promote Proliferation, Migration and Neurotrophin Expression in Schwann Cell-like Adipose Stem Cells, Potentially Improving Peripheral Nerve Regeneration]]> https://www.researchpad.co/article/N303e64b5-42e1-4589-81f7-84d8bc783962 <![CDATA[SAT-735 Effects of Androgen Receptor Activation on Angiogenesis]]> https://www.researchpad.co/article/N88281a86-ab84-4a85-bb9c-3a8754e7b6ff <![CDATA[SAT-742 Characterising the Metabolism, Glucuronidation and Sulfation of C11-oxy C19 Steroids]]> https://www.researchpad.co/article/N864ed796-9945-498e-87c8-17f70dba6baa <![CDATA[SAT-745 Lower Serum Estradiol Levels in Assigned Female at Birth Transgender People with Initiation of Testosterone Therapy: Results from the European Network for the Investigation of Gender Incongruence (ENIGI)]]> https://www.researchpad.co/article/Ncbd4d92b-a522-4f68-9ce0-6c85d5c4cb27

Abstract

Introduction: Aromatization of exogenous testosterone might result in increased estradiol levels. Concerns have been raised about undesired estrogenic effects in assigned female at birth (AFAB) transgender people. How serum estradiol levels change after initiation of testosterone therapy and if these levels should be monitored, remains unclear. Methods: This prospective cohort study was part of the European Network for the Investigation of Gender Incongruence (ENIGI). Serum levels of sex steroids were assessed in 746 AFAB transgender people during a three-year follow-up period, starting at the initiation of hormone treatment. Results: Estradiol levels decreased from median [P25-P75] 45.5[24.0-102.2]pg/mL to 36.5[25.0-46.2]pg/mL over three years (P<0.001), a change was already noticeable during the first three months (mean - 17.1 pg/mL, 95% CI -23.8 - -10.6, P<0.001). Serum estradiol levels were lower in people without endogenous estradiol production (contraceptive users or post gonadectomy) at baseline and after three months, compared to people with endogenous estradiol production. Using long acting testosterone undecanoate injections resulted in a more prominent decrease in serum estradiol values over twelve months, compared to short acting mixed testosterone esters (P<0.001) or testosterone gel (P=0.001). Changes in serum estradiol were positively correlated to changes in LH (ρ = 0.107, P<0.001) and negatively correlated to changes in FSH levels (ρ=-0.167, P<0.001) and body mass index (ρ=-0.082, P<0.001). Conclusion: Testosterone administration in AFAB transgender people results in decreasing serum estradiol levels. Although an underlying mechanism for the observed decrease in serum estradiol levels remains difficult to fathom, our results suggest that testosterone administration suppresses endogenous estradiol production. The exception found in people without endogenous estradiol production may be attributed to aromatization of exogenous testosterone.

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<![CDATA[SAT-744 Spiral Steroid Lactones Are Synthesized by Condensation of a Steroid Precursor with Coenzyme a Derivatives]]> https://www.researchpad.co/article/N3afb12b9-0643-4efa-b15a-d8a7f6db835c

Abstract

: Szent-Gyorgyi proposed that digoxin wasn’t really drug but was a substitute for an endogenous cardiotonic steroid (ECS). Endogenous ouabain and marinobufagenin have been proposed as ECS.

: Ionotropin, our first candidate for the ECS, is unique among steroids because it is the phosphocholine ester of a steroid with 23 carbon atoms. Logically, either there must be a novel mechanism for adding carbon atoms to a pregnenolone-like precursor or a novel mechanism for side-chain cleavage from a cholesterol-like precursor.

: Serum samples were extracted with acetonitrile, filtered and analyzed by MS-

on an LTQ-XL ion trap mass spectrometer. The instrument permits multiple rounds of fragmentation and identification of the parent ion and each fragment ion. This process permitted recognition of ions that were phosphocholine esters and of the mass of the steroid fragments. The chemical formula of each steroid fragment was determined by trial and error analysis. Although not every mass ion has a unique chemical formula, in fact, each of the steroid ions had a unique formula. Possible isomers were resolved by consideration of knowledge of steroid biosynthetic pathways.

: In brief, human serum samples had steroid fragment ions consistent with 23 (354 Da) and 25 (398 Da) carbon atoms. This provides an additional constraint as the synthetic mechanism must account for both products. These mass ions were consistent with condensation of either acetyl-CoA or acetoacetyl-CoA with the phosphocholine ester of pregna-5,7-diene-3β,17α-diol-20-one. After condensation, the steroid adduct would be dehydrated and cyclized to form the corresponding spiral steroid phosphocholine ester. This pathway is similar to the mechanism of addition of 2 carbon fragments to a long chain fatty acid. This is the first explanation for the biosynthesis of endogenous mammalian ECS. Spiral lactones would be expected to cross react with many antibodies specific for digoxin, ouabain or marinobufagenin. Either one of the spiral lactones would satisfy Szent-Gyorgyi’s suggestion as the endogenous digoxin-like material.

: In summary, we have isolated 2 spiral steroid lactones from mammals and identified the mechanism of their biosynthesis. We propose, as the spiral steroids share structural features with the spironolactone class of potassium sparing diuretics, that they also share functions. Nicholls proposed that a candidate for ECS should not be accepted without [a] isolation, [b] precursors, and [c] a biosynthetic path. As there has been no satisfaction of these requirements for ouabain or marinobufagenin, their existence as ECS in mammals needs to be reconsidered.

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<![CDATA[SAT-740 Plasma Glucocorticoids and Mineralocorticoids Are Associated to Metabolic Syndrome Features in Women]]> https://www.researchpad.co/article/N95d51532-f3fa-4eec-9099-d16f7e6c9976

Abstract

: Excess visceral adipose tissue accumulation on anatomical structures such as the greater omentum and mesentery are strong predictors of obesity-associated comorbidities (1). High glucocorticoid levels have been associated with body fat distribution and preferential visceral fat accumulation as well as features of the metabolic syndrome (MetS) (2). These effects are thought to be mediated by the glucocorticoid receptor, a nuclear receptor showing affinity for both glucocorticoids and mineralocorticoids. In this study, we examined plasma concentrations of glucocorticoids and mineralocorticoids in women with or without the MetS. In addition, we assessed the ability of these steroids to predict fat accumulation and features of the MetS.

: In a sample of 49 women (age 47 ± 4.99 years; BMI 26.4 ± 4.70 kg/m

), plasma concentrations of cortisol, 11-deoxycortisol, cortisone, aldosterone, corticosterone and 11-deoxycorticosterone were analyzed by electrospray ionization-liquid chromatography-tandem mass spectroscopy (ESI-LC-MS/MS). Metabolic parameters were assessed to establish the presence of the MetS using NCEP-III criteria. Subcutaneous and visceral adipocyte cell size was measured by histomorphometry.

: We found HDL-triglycerides to be positively associated with levels of 11-deoxycorticosterone, 11-deoxycortisol, corticosterone, cortisone and cortisol (p<0.05 for all). 11-deoxycorticosterone concentration was also negatively associated with waist circumference (-0.294, p<0.05), LDL-cholesterol and LDL-triglyceride content (-0.264 and -0.362, p<0.05) whereas cortisone level was positively associated with fasting glucose (0.3, p<0.05). Our model including mineralocorticoids predicted systolic blood pressure (R

=0.303), while the one including glucocorticoids predicted HDL-cholesterol (R

=0.495). In addition, as expected, we found that women with the MetS were characterized by significantly higher percentage body fat and displayed subcutaneous and visceral adipocyte hypertrophy (p<0.05). Interestingly, women with the MetS also showed a trend for lower plasma cortisol concentrations (p=0.07).

: Our data suggest that glucocorticoids and mineralocorticoids are associated with individual components of the MetS in women.

(1) Tchernof et al.(2013), Physiol Rev, 93(1);

(2) Constantinopoulos et al., (2015), Eur J Endocrinol, 172(1)

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<![CDATA[OR09-04 Common Genetic Variants Associated with SERPINA6 Expression in Liver Influence Cortisol-Responsive Transcriptional Networks in Human Adipose Tissue]]> https://www.researchpad.co/article/N7923ded7-bfe3-4f75-bf3c-b00994f94d12 <![CDATA[SAT-LB134 Aging Related Changes in Sex Hormone-Binding Globulin in Men With HIV]]> https://www.researchpad.co/article/N4cfa2dc4-fb45-42a0-b049-1d9a64c07838 <![CDATA[OR09-05 Expression of SLC35F1 in the Plasma Membrane of Cells of Aldosterone Producing Cell Clusters (APCCs) and Its Possible Role in Aldosterone Synthesis]]> https://www.researchpad.co/article/N95fc7cdc-1de4-421a-b024-965d4f5a38e9 <![CDATA[SAT-738 Sources of Error in Estimation of Cortisol Half-Life Using Conventional, Single-Compartment Model: Bias Due to Variation in CBG Concentration]]> https://www.researchpad.co/article/Ncb13fc8b-b65c-44fc-80e1-8ed4ec9c1303 <![CDATA[SAT-LB136 A Proteomic Approach to Identify Circulating Glucocorticoid Responsive Proteins in Humans]]> https://www.researchpad.co/article/N40e04fdb-24ed-4818-bc8b-66f14bf0e30d