ResearchPad - adrenal-physiology-and-disease Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SUN-219 Electron Transport Chain Complex 2 in Mitochondrial Pregnenolone Synthesis]]> The mitochondrial P450 family of enzymes (SCC), which require the electron transport chain (ETC) complexes III, IV and V, initiate steroidogenesis by cleaving the sidechain of cholesterol to synthesize steroid hormones, an essential component for mammalian survival. SCC is required for full-term gestation, and aberrant expression may cause pseudohermaphroditism, breast cancer or polycystic ovary syndrome. Complex II or succinate dehydrogenase (quinone) is shared with the TCA cycle and has no proton pumping capacity and no known role in steroid synthesis. We now show that succinate is an intermediate metabolite in the TCA cycle and plays a central role physiologically. Specifically, complex II is required for SCC activation, where the proton pump facilitates an active intermediate state conformation at the matrix, so that in the presence of succinate, ATP can add phosphate. A longer intermediate equilibrium state generates a transient stabilization to enhance the binding of phosphate anions in the presence of succinate anions, resulting in higher enthalpy and activity. An inhibition of the processing at the intermediate state stops phosphate addition and activity. We further describe that phosphate circulation brings the molten globule, an intermediate, to an active folded state. This is the first report showing that an intermediate state activated by succinate facilitates ETC complex II interaction with complexes III and IV for metabolism.

<![CDATA[SUN-220 High Salt Intake May Paradoxically Drive Autonomous Aldosterone Production]]> Many modifiable factors contribute to the high prevalence rates of hypertension, among them is the consumption of too much salt (sodium). Another curable cause of hypertension is the excess of the hormone aldosterone. Aldosterone is normally produced by the zona glomerulosa (ZG) of the adrenal glands in response to a lack of salt and conversely suppressed by salt excess. We hypothesize that [i] suppression of aldosterone production induces apoptosis of ZG cells, as occurs following genetic deletion1; [ii] this sets up a maladaptive response to chronic salt overload by conferring a survival advantage to cells in which mutations drive autonomous aldosterone production. To address [i], we measured apoptosis of cells in which aldosterone synthesis was inhibited; to address [ii] we undertook a cross-sectional clinical study of aldosterone and sodium excretion, hypothesising that aldosterone excretion will be highest in the outside quartiles of sodium excretion. Aldosterone was inhibited by modification, in human adrenocortical H295R cells, of either CADM1 expression (mutated in aldosterone-producing adenomas2) or intracellular calcium concentration3. Apoptosis was measured by flow cytometric analysis of annexin V conjugates. 24-hour urinary aldosterone excretion (24h-Ualdo) was correlated with 24-hour urinary sodium (24h-Usodium) in 24h-urine samples collected for a Malaysian population-based salt intake study (MyCoSS). The prevalence of autonomous aldosterone production was estimated from the proportion of subjects with serum measurement whose “SUSSPUP” ratio (= serum sodium to urinary sodium)/(serum potassium2 to urinary potassium) was >5.34. Modification of CADM1 in human adrenocortical H295R cells decreased aldosterone production by half compared to vector control, and this was associated with a 3 to 5-fold increase of apoptotic cells (p<0.05; n>3). Pilot investigation of a Cav1.3 inhibitor decreased aldosterone production by 70%, and increased apoptosis by 7-fold (p<0.10; n=2). In 767 subjects, 24h-urine samples from the high urinary sodium quartile (>150mmol/d) had higher urinary aldosterone (4+0.18 ug/d) than other quartiles (p=0.00001). Overall, the estimated prevalence of autonomous aldossterone production using SUSSPUP ratio was 4.5% (8 of 179 subjects). In 63 subjects with 24h-Usodium>200 mmol/day, autonomous aldosterone secretion (conventionally >10µg/d) was found in 9.5%. Our results support the hypothesis that initial suppression of aldosterone production by salt excess may create a selective advantage for cells which autonomously produce aldosterone, and hence an inappropriate long-term increase in aldosterone production.

1Lee et al., Endocrinology. 2005;146:2650-6.

2Wu et al., 21st European Congress of Endocrinology. Vol. 63. BioScientifica, 2019.

3Xie et al., Sci Rep. 2016;6:24697.

4Willenberg et al., Eur J Clin Invest. 2009;39:43-50.

<![CDATA[SUN-210 Differentially Expressed Mirnas in Zona Reticularis (ZR) Cells of the Human Adrenal Cortex]]> The human adrenal cortex, involved in adaptive responses to stress, fluid homeostasis, and secondary sexual characteristics, arises from a tightly regulated development of a zone and cell type-specific secretory pattern. However, the molecular mechanisms governing adrenal zonation, particularly postnatal ZR development, which produces adrenal androgens in a life-time-specific manner, remain poorly understood. The hallmark of ZR is the low expression of type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). However, the mechanisms underlying HSD3B2 downregulation in the ZR remain unknown. MiRNAs are seen as regulators of cell phenotypes. The objective of the study was to compare miRNA expression profiles in human adrenal ZR and zona fasciculata (ZF). ZF and ZR were microdissected from human adrenals tissues (n=5, from boys aged 16, 17 and 19 yr and girls aged 12 and 16 yr) by laser capture microdissection (LCM). Total RNA was extracted from 5 ZF/ZR pairs and next-generation sequencing (NGS) was used to perform the microRNA expression profiling. 281 mature microRNAs were identified in human adrenal cortex. Among them, 7 microRNAs were significantly differentially expressed between ZF and ZR. The expression of miR-375-3p, miR-483-3p and miR-7-5p was higher in ZR compared with its paired ZF by 2-fold or greater. Multiple available bioinformatic algorithms (TargetScan, miRanda, DianaLab and PicTar) were employed to search for its target genes. Among predicted target genes, several genes (GATA-6, GATA-4, SF1, NR4A2, and IGF-1) are known to be involved in HSD3B2 regulation. In summary, LCM combined with NGS provided a robust approach to explore the adrenal zone-specific micro-RNA profiling. Our data gave first hints that miRNAs might be novel regulatory modules associated with human adrenal ZR cell-specific transcript regulation underlying developmental androgen production.

<![CDATA[SUN-218 Analysis of ATRX and ZNRF3 Expression and Copy Number Variation in a Pediatric and Adult Cohort with Adrenocortical Tumors]]> Introduction: Adrenocortical carcinoma (ACC) is a rare malignancy with an incidence of 1 to 2 cases per million/year in adults and with a global survival rate of less than 40% in 5 years. ACC diagnosis was based on Weiss criteria for adult patients. Pediatric patients with adrenocortical tumors (PAT), in general, are associated with better survival in most cases, and the malignant disease is established when local or distant metastases are found. The integrated and extensive genomic-molecular characterization of ACC has resulted in a better understanding of its pathophysiology. Some studies have demonstrated the involvement of ATRX and ZNRF3 genes in adrenal tumorigenesis in pediatric and adult patients with PAT and ACC, respectively. However, these data have not been validated in a Brazilian cohort with a high prevalence of the TP53 germline R337H mutation. Objectives: We aimed to evaluate the ATRX and ZNRF3 expression and copy number variation in a Brazilian cohort of patients with PAT and adults with ACC from a tertiary center. Patients and Methods: 34 adults (19 women - 56%) with median age 49 years old (range 18-83) and twelve pediatric patients (7 girls - 58%) with median age three years old (0.8-15 years old) were included in this study. The epidemiological data, clinical presentation, hormonal data, radiological imaging, and genetic background for TP53 were retrospectively evaluated. MLPA and RT-PCR were employed to evaluate the copy number variation and the gene expression, respectively, of ATRX and ZNRF3 in tumor tissues. Results: Adult group: Seven patients out of 27 (25.9%) presented the pathogenic germline mutation pR337H onTP53. 20 patients (58.8%) presented metastasis, and 19 (55%) had a fatal outcome. The median global survival was 17.23 months (0.6-185.8 m). Pediatric group: 10 patients (83.3%) presented the pathogenic germline mutation p.R337H on TP53. Four patients presented metastasis and only two had a fatal outcome. The median global survival was 42.4months (6.63-125.5m). All tumors were functional. Molecular results: Three out of 33 adult patients (9%), and 2 out of 12 (16.6%) pediatric patients presented deletion on ATRX. Four out of 25 adults (16%) and 2 out of 12 pediatric (16.6%) patients showed deletion on ZNRF3. There was no correlation between ATRX and ZNRF3 expressions or deletions with the overall survival rate (p>0.05). The decrease in the ATRX expression was associated with the presence of TP53 germline mutation in pediatric and adult cohorts (p 0.028). Conclusion: We confirmed the presence of alterations on ATRX and ZNRF3 genes in both cohort (adult and pediatric tumors). These results differ from the previous studies, which demonstrated ATRX and ZNRF3 alterations were present in pediatric and adult tumors, respectively. However, prospective studies with larger cohorts are necessary to confirm the prognostic value of ATRX and ZNRF3 genes in PAT and adults with ACC.

<![CDATA[SUN-214 A Novel Glucocorticoid Receptor Antagonist (CORT113176) Reveals Unique Developmental and Tissue-Specific Effects in a Neonatal Rat Model of Human Prematurity]]> <![CDATA[SUN-211 Active Steroid Hormone Synthesis Renders Adrenocortical Cells Highly Susceptible to Type II Ferroptosis Induction]]> <![CDATA[SUN-212 Interference in Serum Androstenedione Measured by LC-MS/MS in Newborns Samples]]> <![CDATA[SUN-222 Pre- and Post-Pubertal Reference Ranges for Oxygenated Androgens in Saliva]]> <![CDATA[SUN-208 The Intra-Individual Variability of 11-Ketotestosterone and 11β-Hydroxyandrostenedione]]> <![CDATA[SUN-LB41 The Role of Androgen Receptors in Female Mouse X-Zone Loss During Aging and Pregnancy]]> <![CDATA[SUN-LB43 DLK1 Expressing Cells Contribute to the Zonation of the Adrenal Gland]]> <![CDATA[SUN-LB42 The Sexually Dimorphic Response of the Mouse Adrenal Inner Cortex to Thyroid Hormone Treatment]]> <![CDATA[SUN-213 The Role of Filamin A (FLNA) in the Regulation of Insulin-Like Growth Factor System in Adrenocortical Carcinomas]]> <![CDATA[SUN-LB39 ATAC-Seq Reveals Dynamic Changes in Chromatin Accessibility Following PKA Activation in NCI-H295R Adrenocortical Cells]]> <![CDATA[SUN-216 Investigating the Role of the Liver X Receptor in Potentiating Mitotane Therapy in Adrenocortical Carcinoma]]> <![CDATA[SUN-221 Subclinical Alpha-1 Antitrypsin Deficiency Is Associated with Increased Free Cortisol Fraction in Plasma and Altered Glucocorticoid Delivery to Tissues]]> 85% of plasma cortisol and controls the circulating free cortisol pool. Proteolytic cleavage by neutrophil elastase is proposed to reduce CBG binding affinity and increase free cortisol availability to inflamed tissues. The CORtisol NETwork (CORNET) consortium found that genetic variation at a locus spanning SERPINA1 (encoding alpha-1 antitrypsin, A1AT, the endogenous inhibitor of neutrophil elastase) and SERPINA6 (CBG) contributes to morning total plasma cortisol variation. We hypothesised that A1AT deficiency increases CBG cleavage and hence free plasma cortisol, resulting in increased tissue cortisol delivery in adipose and in HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1.Methods16 healthy carriers of one of the two most common A1AT-deficiency single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. Participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 400mg and spironolactone 200mg, or placebo in a double blind randomised crossover design. Plasma free cortisol was measured by isotopic dilution and ultrafiltration, total cortisol by LC-MS/MS, total CBG by ELISA, CBG binding capacity by radioligand displacement assay, and ACTH by immunoassay. Serum A1AT was measured by ELISA. Tissue cortisol (LC-MS/MS) and expression of glucocorticoid dependent transcripts (qPCR) were measured in subcutaneous adipose samples collected by needle biopsy.ResultsSerum A1AT was confirmed lower in those with heterozygous mutations vs wild type controls (411.3 +/- 27.44 vs 565.1 +/- 23.38 mg/dL, p=0.0002). No measurable differences in total CBG or CBG binding capacity were observed. However, plasma free cortisol fraction was higher in those carrying A1AT mutations (16.13 +/- 0.2 vs 13.88 +/- 0.04 %, p<0.0001). Adipose cortisol concentrations were not significantly different but expression of glucocorticoid responsive genes e.g. PER1 was 54% higher (p=0.014) in A1AT-deficient subjects. Plasma cortisol was elevated during CRASH testing in both groups, with the increment versus placebo tending to be lower in A1AT-deficient subjects (82.5 +/- 6.7 vs 126.7 +/- 6.8 nM).ConclusionAlpha-1 antitrypsin mutation heterozygosity, common in the general population, is associated with higher free cortisol fraction, consistent with enhanced cleavage of CBG. This is associated with evidence of enhanced delivery of glucocorticoid to adipose tissues but reduced HPA negative feedback, suggesting tissue-specific control of cortisol delivery by CBG. ]]> <![CDATA[SUN-LB40 Chronic Cortisol Works Through the Transcription Factor KLF9 to Deregulate Immune Response and Metabolism]]> <![CDATA[SUN-217 Skin Glucocorticoid Metabolism in Burn Injury: Towards Novel Treatments That Reduce Scarring]]> <![CDATA[SUN-207 Modelling Long Term Glucocorticoid-Induced HPA Axis Suppression in Mice]]>


Glucocorticoids are prescribed for >3 months to 1% of the UK population. 10-50% of these long-term glucocorticoid treated patients develop persistent HPA axis suppression associated with mortality and morbidity. We have developed a mouse model of glucocorticoid-induced HPA axis dysfunction to determine the mechanisms resulting in persistent HPA axis suppression.

36 C57BL/6 adult male mice received Dexamethasone (DEX,~10µg/day) or vehicle (CTL) via drinking water for 28 days, following which treatment was stopped and tissues were harvested at 0, 7 and 28 days. DEX suppressed waking serum corticosterone at days 0 and 7, recovering by day 28. Adrenal size remained lower 28 days following DEX withdrawal. DEX had no effect on whole pituitary


expression, although

was increased at day 0. In the adrenal,


expression were reduced at time 0; normalising by 28 days.

24 POMC-GFP male mice were treated as above. Tissues were collected at day 0 (n=6), 7 (n=3) and 10 (n=3) following withdrawal. Pooled corticotrophs (groups of 3) were isolated by FACS and RNA extracted for qPCR. DEX reduced corticotroph

expression at time 0 (x20 fold reduction), with x5 fold suppression at day 7, which recovered with evidence of compensation by day 10. DEX increased expression of

but not


CONCLUSION: 28 days dexamethasone treatment in mice suppresses the HPA axis. HPA suppression is evident 7 days following withdrawal of dexamethasone in the adrenal, corticotroph population and corticosterone production. Further analysis will determine mechanisms for delays in HPA axis recovery.