Comparative Assessment of the Influence of Embolizing Agents on the Adrenal Parenchyma.

A comparative analysis of morphological and functional changes in the adrenal glands after embolization of supplying arteries with Lifepearl microspheres (100±25 µm; Terumo) and with a solution of polidocanol (Aethoxysklerol, Kreussler Pharma) was performed in male Vietnamese Lop-Bellied pigs (n=20). The introduction of the microspheres into the arterial bed did not affect the adrenal parenchyma. However, the injection of the liquid sclerosing agent caused foci of productive inflammation leading to the formation of sclerotic tissue and reduction of the volume of glandular tissue. Administration of the sclerosant was accompanied by arterial hypotension, an indirect indication of a decrease in vasoactive hormone production by the adrenal cortex. These findings suggest that polidocanol can be used as an embolization agent for hyperplastic diseases of the adrenal cortex.

Vitamin D3 application and factors of its activity in the adrenal cortex of andropausal rats: A functionally-histological study.

INTRODUCTION AND AIM: Vitamin D supplementation in aging subjects manifests a positive effect on various health-related parameters. We performed a functionally-histological analysis of the adrenal cortex regarding the factors of vitamin D activity and corticosterone output after vitamin D3 application in a rat model of the andropause. MATERIAL AND METHODS: Middle-aged Wistar rats were divided into sham operated (SO; n=8), orchidectomized (Orx; n=8) and vitamin D3-treated orchidectomized (Orx+vit. D; n=8) groups. Vitamin D3 (5 µg/kg b.m.) was administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using histochemistry/immunohistochemistry, stereology, ultrastructural and biochemical analyses. RESULTS: Orchidectomy (Orx) decreased the adrenal cortex-related volume densities of vascular (p<0,0001), vitamin D receptor (VDR; p<0,0166), cytochrome P450 oxidase 2R1 (CYP 2R1; p<0,0001) and cytochrome P450 oxidase 24 (CYP 24; p<0,0001) depots, but increased the volume density of cytochrome P450 27B1 (CYP 27B1; p<0,0001) depots. In Orx+vit. D rats, increase of the adrenal cortex-related volume densities of collagen (p<0,0001), VDR (p<0,0001) and CYP 2R1 (p<0,0001) depots as well as the lipid-droplet diameter (p<0,0001) in adrenocortical outer zona fasciculata cells was observed, while a decrease of volume densities of the vascular (p<0,0001), CYP 27B1 (p<0,0001) and CYP 24 (p<0,0001) depots was registered, all versus Orx group. Plasma level of ACTH was decreased (p=0,0155) and serum concentrations of 25-hydroxyvitamin D3 and corticosterone were increased (p<0,0001 and p=0,0187, respectively), all after the same treatment. CONCLUSIONS: Increased corticosterone output after vitamin D3 application to andropausal rats appears not to be related to increased availability of 25-hydroxyvitamin D3 and decreased degradation of 1,25-dihydroxyvitamin D3 in adrenal tissue, but rather involves the central regulatory mechanisms.

Increased adrenocortical activity in patients with vasovagal syncope.

OBJECTIVE: Syncope is a transient loss of consciousness resulting from cerebral hypoperfusion. Vasovagal syncope (VVS) is a form of orthostatic intolerance (OI). Its clinical signs such as dizziness and hypotension may mimic symptoms of adrenal insufficiency. The objective of this study was to evaluate the adrenal gland function in patients with vasovagal syncope after stimulation with synthetic adrenocorticotropic hormone (ACTH). DESIGN: Case-control study on patients with VVS and healthy controls. METHODS: The study involved 42 participants, including 27 patients diagnosed with VVS using the head-up tilt test and 15 healthy individuals with no history of syncope or any orthostatic symptoms. Serum cortisol and aldosterone concentrations were measured under basal conditions and at 30 and 60 min after intramuscular ACTH stimulation. RESULTS: Patients with VVS had significantly higher cortisol levels at baseline (441 ± 143 vs. 331 ± 84.7 nmol/L, p = 0.01), at 30 min (802 ± 143 vs. 686 ± 105 nmol/L, p = 0.01) and at 60 min (931 ± 141 nmol/L vs. 793 ± 147 nmol/L, p = 0.001) after ACTH administration (Synacthen 250 µg). Plasma aldosterone increased after ACTH stimulation, but did not show significant differences among groups. Furthermore, there was also no significant correlation between cortisol levels and blood pressure or heart rate. CONCLUSION: Patients diagnosed with VVS have higher cortisol levels both at baseline and after ACTH stimulation. This finding indicates that individuals with VVS have higher adrenocortical activity potentially as a response to the orthostatic stress induced by syncope, which acts as a stressful stimulus on the autonomic nervous system.

Disruptive effects of plasticizers bisphenol A, F, and S on steroidogenesis of adrenocortical cells.

Introduction: Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). Methods: NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Results: Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. Discussion: In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.

Increased adrenal steroidogenesis and suppressed corticosteroid responsiveness in critical COVID-19.

BACKGROUND: The effect of coronavirus disease 2019 (COVID-19) on adrenal endocrine metabolism in critically ill patients remains unclear. This study aimed to investigate the alterations in adrenal steroidogenic activity, elucidate underlying mechanisms, provide in situ histopathological evidence, and examine the clinical implications. METHODS: The comparative analyses of the adrenal cortices from 24 patients with fatal COVID-19 and 20 matched controls were performed, excluding patients previously treated with glucocorticoids. SARS-CoV-2 and its receptors were identified and pathological alterations were examined. Furthermore, histological examinations, immunohistochemical staining and ultrastructural analyses were performed to assess corticosteroid biosynthesis. The zona glomerulosa (ZG) and zona fasciculata (ZF) were then dissected for proteomic analyses. The biological processes that affected steroidogenesis were analyzed by integrating histological, proteomic, and clinical data. Finally, the immunoreactivity and responsive genes of mineralocorticoid and glucocorticoid receptors in essential tissues were quantitatively measured to evaluate corticosteroid responsiveness. FINDINGS: The demographic characteristics of COVID-19 patients were comparable with those of controls. SARS-CoV-2-like particles were identified in the adrenocortical cells of three patients; however, these particles did not affect cellular morphology or steroid synthesis compared with SARS-CoV-2-negative specimens. Although the adrenals exhibited focal necrosis, vacuolization, microthrombi, and inflammation, widespread degeneration was not evident. Notably, corticosteroid biosynthesis was significantly enhanced in both the ZG and ZF of COVID-19 patients. The increase in the inflammatory response and cellular differentiation in the adrenal cortices of patients with critical COVID-19 was positively correlated with heightened steroidogenic activity. Additionally, the appearance of more dual-ZG/ZF identity cells in COVID-19 adrenals was in accordance with the increased steroidogenic function. However, activated mineralocorticoid and glucocorticoid receptors and their responsive genes in vital tissues were markedly reduced in patients with critical COVID-19. INTERPRETATION: Critical COVID-19 was characterized by potentiated adrenal steroidogenesis, associated with increased inflammation, enhanced differentiation and elevated dual-ZG/ZF identity cells, alongside suppressed corticosteroid responsiveness. These alterations implied the reduced effectiveness of conventional corticosteroid therapy and underscored the need for evaluation of the adrenal axis and corticosteroid sensitivity.

Effects of androgenic modulation on the morphophysiology of the adrenal cortex of male gerbils.

The study aimed to investigate the repercussions of androgen modulation on the adrenal cortex of male gerbils, focusing on the morphophysiology, proliferation, and cell death, as well as the expression of hormone receptors and steroidogenic enzymes. Mongolian gerbils (Meriones unguiculatus) were divided into three experimental groups: Control (C), Testosterone (T), animals received injections of testosterone cypionate and Castrated (Ct), animals underwent orchiectomy. The results showed that castration increased the zona fasciculata and promoted cell hypertrophy in all zones. Testosterone supplementation increased cell proliferation and cell death. Androgen modulation promoted an increase in AR, Erα, and ERß. Castration promoted an increase in the CYP19, while decreasing 17ßHSD enzymes. Testosterone supplementation, on the other hand, reduced CYP17 and increased CYP19 and 3ßHSD enzymes. By analyzing the effects of androgen supplementation and deprivation, it can be concluded that testosterone is responsible for tissue remodeling in the cortex, regulating the rate of cell proliferation and death, as well as cell hypertrophy. Testosterone also modulate steroid hormone receptors and steroidogenic enzymes, consequently affecting the regulation, hormone synthesis and homeostasis of this endocrine gland.

Melatonin counteracts polyethylene microplastics induced adreno-cortical damage in male albino rats.

There are various substances that can disrupt the homeostatic mechanisms of the body, defined as endocrine-disrupting chemicals (EDCs). The persistent nature of microplastics (MPs) is a cause for concern due to their ability to accumulate in food chains and widespread use, making their toxic effects particularly alarming. The potential of MPs for disrupting the endocrine system was observed in multiple tissues. Moreover, the adrenal gland is known to be extremely sensitive to EDCs, while with the effect of MPs on the adrenal gland has not previously been studied. This study aimed to highlight the potential polyethylene microplastics (PE-MPs) induced adreno-toxic effects rather than exploring the implicated mechanisms and concluding if melatonin (Mel) can afford protection against PE-MPs induced adreno-toxicity. To fulfill the goal, six groups of rats were used; control, Mel, PE-MPs (3.75 mg/kg), PE-MPs (15 mg/kg), PE-MPs (3.75 mg/kg) +Mel, and PE-MPs (15 mg/kg) +Mel. PE-MPs induced toxic changes in the adrenal cortex, which was evident by increased adrenal weight, histopathological examination, and ultrastructural changes detected by electron microscope. A reduction in serum cortisol and an increase in serum adrenocorticotropic hormone resulted from the adreno-toxic effects of PE-MPs. Mechanisms may include the reduction of steroidogenesis-related genes, as PE-MPs drastically reduce mRNA levels of StAR, Nr0b1, Cyp11A1, as well as Cyp11B1. Also, oxidative stress that results from PE-MPs is associated with higher rates of lipid peroxidation and decreased superoxide dismutase and glutathione. PE-MPs inflammatory effect was illustrated by elevated expression of IL-1ß and NF-kB, detected by immunohistochemical staining, in addition to increased expression of caspase-3 and mRNA of Bax, markers of proapoptotic activity. The impacts of PE-MPs were relatively dose-related, with the higher dose showing more significant toxicity than the lower one. Mel treatment was associated with a substantial amelioration of PE-MPs-induced toxic changes. Collectively, this study fills the knowledge gap about the MPs-induced adrenal cortex and elucidates various related toxic mechanisms. It also supports Mel's potential protective activity through antioxidant, anti-inflammatory, anti-apoptotic, and gene transcription regulatory effects.

Interrogating the estrogen-mediated regulation of adrenocortical Klotho expression using ovariectomized albino rat model exposed to repeated restraint stress.

Reproductive aging is associated with altered stress response and many other menopausal symptoms. Little is known about the adrenal expression of the anti-aging protein Klotho or how it is modulated by estrogen in ovariectomized stressed rats. Fifty-six Wistar female rats were assigned into seven equal groups. Sham-operated (Sham), sham stressed (Sham/STS), ovariectomized (OVR), ovariectomized stressed (OVR/STS), ovariectomized stressed rosiglitazone-treated (OVR/STS/R), ovariectomized stressed estrogen-treated (OVR/STS/E), and ovariectomized stressed estrogen/GW9662 co-treated (OVR/STS/E/GW) groups. All stressed rats were subjected daily to a one-hour restraint stress test for 19 days. At the end of the experiment, blood was collected for serum corticosterone (CORT) analysis. Adrenal tissues were obtained and prepared for polymerase chain reaction (PCR) assay, hematoxylin and eosin (H&E), immunohistochemistry-based identification of Klotho and PPAR-γ, and Oil Red O (ORO) staining. The rise in serum CORT was negligible in the OVR/STS group, in contrast to the Sham/STS group. The limited CORT response in the former group was restored by estrogen and rosiglitazone and blocked by estrogen/GW9226 co-administration. ORO-staining revealed a more evident reduction in the adrenal fat in the OVR/STS group, which was reversed by estrogen and counteracted by GW. Also, there was a comparable expression pattern of Klotho and PPAR-γ in the adrenals. The adrenal Klotho decreased in the OVR/STS group, but was reversed by estrogen treatment. GW9226/estrogen co-treatment interfered with the regulatory effect of estrogen on Klotho. The study suggested modulation of the adrenal Kotho expression by estrogen, in the ovariectomized rats subjected to a restraint stress test. This estrogen-provided adrenal protection might be mediated by PPAR-γ activation.

Impact of systemic treatments for advanced thyroid cancer on the adrenal cortex.

Background: Fatigue is a frequent adverse event during systemic treatments for advanced thyroid cancer, often leading to reduction, interruption, or discontinuation. We were the first group to demonstrate a correlation between fatigue and primary adrenal insufficiency (PAI). Aim: The objective was to assess the entire adrenal function in patients on systemic treatments. Methods: ACTH, cortisol and all the hormones produced by the adrenal gland were evaluated monthly in 36 patients (25 on lenvatinib, six on vandetanib, and five on selpercatinib). ACTH stimulation tests were performed in 26 cases. Results: After a median treatment period of 7 months, we observed an increase in ACTH values in 80-100% of patients and an impaired cortisol response to the ACTH test in 19% of cases. Additionally, dehydroepiandrosterone sulphate, ∆-4-androstenedione and 17-OH progesterone levels were below the median of normal values in the majority of patients regardless of the drug used. Testosterone in females and oestradiol in males were below the median of normal values in the majority of patients on lenvatinib and vandetanib. Finally, aldosterone was below the median of the normal values in most cases, whilst renin levels were normal. Metanephrines and normetanephrines were always within the normal range. Replacement therapy with cortisone acetate improved fatigue in 14/17 (82%) patients with PAI. Conclusion: Our data confirm that systemic treatments for advanced thyroid cancer can lead to impaired cortisol secretion. A reduction in the other hormones secreted by the adrenal cortex has been first reported and should be considered in the more appropriate management of these fragile patients.

Effects of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients: a double-blind randomized controlled trial.

Etomidate has been advocated to be used in anesthesia for the elderly and the critically ill patients due to its faint effect on cardiovascular system. But the dose-dependent suppression of etomidate on adrenal cortex function leads to the limitation of its clinical application. Clinical research showed that dexmedetomidine could reduce the dose requirements for intravenous or inhalation anesthetics and opioids, and the hemodynamics was more stable during the operation. The objective was to observe the effect of etomidate combined with dexmedetomidine on adrenocortical function in elderly patients. 180 elderly patients scheduled for elective ureteroscopic holmium laser lithotripsy were randomly allocated to PR group anesthetized with propofol-remifentanil, ER group anesthetized with etomidate-remifentanil, and ERD group anesthetized with dexmedetomidine combined with etomidate-remifentanil. Patients in each group whose operation time was less than or equal to 1 h were incorporated into short time surgery group (PR1 group, ER1 group and ERD1 group), and whose surgical procedure time was more than 1 h were incorporated into long time surgery group (PR2 group, ER2 group and ERD2 group). The primary outcome was the serum cortisol and ACTH concentration. The secondary outcomes were the values of SBP, DBP, HR and SpO2, the time of surgical procedure, the dosage of etomidate and remifentanil administered during surgery, the time to spontaneous respiration, recovery and extubation, and the duration of stay in the PACU. The Serum cortisol concentration was higher at t1~2 in ERD1 group compared to ER1 group (P < 0.05). The Serum cortisol concentration at t1~3 was higher in ERD2 group than in ER2 group (P < 0.05). The Serum ACTH concentration was lower at t1~2 in ERD1 group compared to ER1 group (P < 0.05). The Serum ACTH concentration at t1~3 was lower in ERD2 group compared to ER2 group (P < 0.05). The SBP at T1 and T3 were higher in ER2 and ERD2 group than in PR2 group (P < 0.05). The DBP in ER1 and ERD1 group were higher at T1 compared to PR1 group (P < 0.05). The dosage of etomidate was significantly lower in ERD1 group and ERD2 group than in ER1 group and ER2 group (P < 0.05), respectively. The administration of dexmedetomidine combined with etomidate can attenuate the inhibition of etomidate on adrenocortical function in elderly patients and maintain intraoperative hemodynamic stability.

Cis-bifenthrin inhibits cortisol and aldosterone biosynthesis in human adrenocortical H295R cells via cAMP signaling cascade.

Cis-bifenthrin (cis-BF) is a common-used pyrethroid insecticide frequently detected in environmental and biological matrices. Mounting evidence highlights the endocrine disrupting effects of cis-BF due to anti-estrogenic or anti-androgenic activity. However, little is known about the exposure effects of cis-BF on adrenal cortex function. In this study, effects of cis-BF on biosynthesis of adrenal steroids, as well as the potential mechanisms were investigated in human adrenocortical carcinoma (H295R) cells. Cis-BF decreased basal production levels of cortisol and aldosterone, as well as cAMP-induced production of cortisol. Both he basal and cAMP-stimulated transcriptional levels of several steroidogenic genes were significantly down-regulated by cis-BF. As an important rate-limiting enzyme in steroidogenesis, the protein level of StAR was prohibited by cis-BF on both basal and cAMP-induced conditions. Intracellular level of cAMP was significantly reduced by cis-BF. Overall, these data suggest that cis-BF may inhibit the biosynthesis of cortisol and aldosterone via disrupting cAMP signaling cascade.

A Novel Etomidate Analog EL-0052 Retains Potent Hypnotic Effect and Stable Hemodynamics without Suppressing Adrenocortical Function.

Etomidate is a potent and rapidly acting anesthetic with high therapeutic index (TI) and superior hemodynamic stability. However, side effect of suppressing adrenocortical function limits its clinical use. To overcome this side effect, we designed a novel etomidate analog, EL-0052, aiming to retain beneficial properties of etomidate and avoid its disadvantage of suppressing adrenocortical steroid synthesis. Results exhibited that EL-0052 enhanced GABAA receptors currents with a concentration for EC50 of 0.98 ± 0.02 µM, which was about three times more potent than etomidate (3.07 ± 1.67 µM). Similar to hypnotic potency of etomidate, EL-0052 exhibited loss of righting reflex with ED50s of 1.02 (0.93-1.20) mg/kg in rats and 0.5 (0.45-0.56) mg/kg in dogs. The TI of EL-0052 in rats was 28, which was higher than 22 of etomidate. There was no significant difference in hypnotic onset time, recovery time, and walking time between EL-0052 and etomidate in rats. Both of them had minor effects on mean arterial pressure in dogs. EL-0052 had no significant effect on adrenocortical function in dogs even at a high dose (4.3 × ED50), whereas etomidate significantly inhibited corticosteroid secretion. The inhibition of cortisol synthesis assay showed that EL-0052 had a weak inhibition on cortisol biosynthesis in human H259 cells with an IC50 of 1050 ± 100 nM, which was 2.09 ± 0.27 nM for etomidate. EL-0052 retains the favorable properties of etomidate, including potent hypnotic effect, rapid onset and recovery, stable hemodynamics, and high therapeutic index without suppression of adrenocortical function. SIGNIFICANCE STATEMENT: The novel etomidate analog EL-0052 retains the favorable properties of etomidate without suppressing adrenocortical function and provides a new strategy to optimize the structure of etomidate.

Triadimefon suppresses fetal adrenal gland development after in utero exposure.

Triadimefon is a broad-spectrum antifungal agent, which is widely used in agriculture to control mold and fungal infections. It is considered an endocrine disruptor. Whether triadimefon exposure can inhibit the development of fetal adrenal glands and the underlying mechanism remain unclear. Thirty-two pregnant female Sprague-Dawley rats were randomly divided into four groups. Dams were gavaged triadimefon (0, 25, 50, and 100 mg/kg/day) daily for 10 days from gestational day (GD) 12 to GD 21. Triadimefon significantly reduced the thickness of the zona fasciculata of male fetuses at 100 mg/kg, although it did not change the thickness of the zona glomerulosa. It significantly reduced the serum aldosterone levels of male fetuses at a dose of 100 mg/kg, and significantly reduced serum corticosterone and adrenocorticotropic hormone levels at doses of 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of Agtr1, Mc2r, Star, Cyp11b1, Cyp11b2, Igf1, Nr5a1, Sod2, Gpx1, and Cat, but did not affect the mRNA levels of Scarb1, Cyp11a1, Cyp21, Hsd3b1, and Hsd11b2. Triadimefon markedly reduced AT1R, CYP11B2, IGF1, NR5A1, and MC2R protein levels. Triadimefon significantly reduced the phosphorylation of AKT1 and ERK1/2 at 100 mg/kg without affecting the phosphorylation of AKT2. In contrast, it significantly increased AMPK phosphorylation at 100 mg/kg. In conclusion, exposure to triadimefon during gestation inhibits the development of fetal adrenal cortex in male fetuses. This inhibition is possibly due to the reduction of several proteins required for the synthesis of steroid hormones, and may be involved in changes in antioxidant contents and the phosphorylation of AKT1, ERK1/2, and AMPK.

Adrenal Corticosteroid Perturbation by the Endocrine Disruptor BDE-47 in a Human Adrenocortical Cell Line and Male Rats.

Polybrominated diphenyl ethers (PBDEs) have been previously shown to alter various endocrine biosynthetic pathways. Growing epidemiological evidence suggests that PBDEs alter cardiovascular function. The goal of this study was to examine the effects of BDE-47 on adrenal corticosteroid pathways that play vital roles in cardiovascular homeostasis and pathophysiology. The effect of BDE-47 on aldosterone and cortisol secretion was characterized in a human adrenocortical cell line. HAC15 cells were exposed to various concentrations of BDE-47 (1 nM to 100 µM). Cell viability, corticosteroid secretion, gene expression of enzymes involved in corticosteroid synthesis, and metabolic activity was examined. Additionally, Sprague Dawley male rats were orally exposed to BDE-47 (10 or 100 µg/kg), 5 days per week for 16 weeks. Organ weights and plasma corticosteroid levels were measured. In HAC15 cells, basal and stimulated aldosterone and cortisol secretion was significantly increased by BDE-47. Gene expression of several enzymes involved in corticosteroid synthesis and mitochondrial metabolism also increased. In Sprague Dawley rats, adrenal but not heart, kidney, or liver weights, were significantly increased in BDE-47 treatment groups. Plasma corticosterone levels were significantly increased in the 100 µg BDE-47/kg treatment group. No change in plasma aldosterone levels were observed with BDE-47 exposure. These data indicate that BDE-47 disrupts the regulation of corticosteroid secretion and provides further evidence that PBDEs are potential endocrine disruptors. Future studies will determine the underlying molecular mechanism of altered corticosteroid production and examine whether these alterations result in underlying cardiovascular disease in our rodent model of 16-week BDE-47 exposure.

Functional remodeling of adrenal steroidogenic tissue by food deprivation in the lizard, Sceloporus undulatus.

The present study examined how food availability interacts with age to modulate lizard adrenal steroidogenic function at the cellular level. Adult male and juvenile male and female Eastern Fence Lizards (Sceloporus undulatus) underwent a period of food deprivation with or without a shorter re-feeding period. Lizards maintained on a full feeding regimen served as the controls. Across the feeding regimens, plasma corticosterone of adult lizards was unchanged whereas that of food-deprived juvenile lizards was increased nearly 7 times and this increase was normalized by a short re-feeding period. Freshly dispersed adrenocortical cells derived from these lizards were incubated with ACTH and the production of selected steroids was measured by highly specific radioimmunoassay. Net maximal steroid rates of juvenile cells were 161% greater than those of adult cells. Adult and juvenile progesterone rates were consistently suppressed by food deprivation (by nearly 48%) and were normalized by a re-feeding period, whereas divergent effects were seen with corticosterone and aldosterone rates. Food deprivation suppressed corticosterone rates of adult cells by 43% but not those of juvenile cells. In a reciprocal manner, food deprivation had no significant effect on aldosterone rates of adult cells, but it suppressed those of juvenile cells by 52%. A short re-feeding period normalized most rates in both adult and juvenile cells and further augmented the adult aldosterone rate by 54%. The effect of the feeding regimens on ACTH sensitivity varied with life stage and with steroid. The overall sensitivity of adult cells to ACTH was nearly three times that of juvenile cells. Collectively, the data presented here and data from previous work indicate that food restriction/deprivation in Sceloporus lizard species causes a functional remodeling of the adrenocortical tissue. Furthermore, life stage adds more complexity to this remodeling.

Comparative proteomic analysis to identify the novel target gene of angiotensin II in adrenocortical H295R cells.

Angiotensin II (Ang II) is a well-known peptide that maintains the balance of electrolytes in the higher vertebrates. Ang II stimulation in the adrenal gland induces the synthesis of mineralocorticoids, mainly aldosterone, through the up-regulation of aldosterone synthase (CYP11B2) gene expression. Additionally, it has been reported that Ang II activates multiple signaling pathways such as mitogen-activated protein kinase (MAPK) and Ca2+ signaling. Although Ang II has various effects on the cellular signaling in the adrenal cells, its biological significance, except for the aldosterone synthesis, is still unclear. In this study, we attempted to search the novel target gene(s) of Ang II in the human adrenal H295R cells using a proteomic approach combined with stable isotopic labeling using amino acid in cell culture (SILAC). Interestingly, we found that Ang II stimulation elevated the expression of phosphofructokinase type platelet (PFKP) in both protein and mRNA levels. Moreover, transactivation of PFKP by Ang II was dependent on extracellular-signal-regulated kinase (ERK) 1/2 activation. Finally, we observed that Ang II treatment facilitated glucose uptake in the H295R cells. Taken together, we here identified PFKP as a novel target gene of Ang II, indicating that Ang II not only stimulates steroidogenesis but also affects glucose metabolism.

Involvement of clock gene expression, bone morphogenetic protein and activin in adrenocortical steroidogenesis by human H295R cells.

Functional interactions between the levels of clock gene expression and adrenal steroidogenesis were studied in human adrenocortical H295R cells. Fluctuations of Bmal1, Clock, Per2 and Cry1 mRNA levels were found in H295R cells treated with forskolin (FSK) in a serum-free condition. The changes of clock gene expression levels were diverged, with Clock mRNA level being significantly higher than Cry1 and Per2 mRNA levels after 12-h stimulation with FSK. After FSK induction, mRNA levels of StAR and CYP11B2 were highest at 12 hours and CYP17 mRNA level reached a peak at 6 hours, but HSD3B1 mRNA level was transiently decreased at 3 hours. The expression levels of Clock mRNA showed a significant positive correlation with StAR among the interrelationships between mRNA levels of key steroidogenic factors and clock genes. Knockdown of Clock gene by siRNA led to a significant reduction of FSK-induced expression of StAR and CYP17 after 12-h treatment with FSK. BMP-6 and activin, which modulate adrenal steroidogenesis, had inhibitory effects on Clock mRNA expression, whereas treatment with follistatin, a binding protein of activin, increased Clock mRNA levels in the presence of FSK, suggesting an endogenous function of activin in regulation of Clock mRNA expression. Collectively, the results indicated that changes of Clock mRNA expression, being upregulated by FSK and suppressed by BMP-6 and activin, were tightly linked to StAR expression by human adrenocortical cells.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Adropin Stimulates Proliferation and Inhibits Adrenocortical Steroidogenesis in the Human Adrenal Carcinoma (HAC15) Cell Line.

Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-ß type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-ß signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.