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The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis -aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.

Adequate oxygen levels are essential for the functioning and maintenance of biological processes in virtually every cell, albeit based on specific need. Thus, any change in oxygen pressure leads to modulated activation of the hypoxia pathway, which affects numerous physiological and pathological processes, including hematopoiesis, inflammation, and tumor development. The Hypoxia Inducible Factors (HIFs) are essential transcription factors and the driving force of the hypoxia pathway; whereas, their inhibitors, HIF prolyl hydroxylase domain (PHDs) proteins are the true oxygen sensors that critically regulate this response. Recently, we and others have described the central role of the PHD/HIF axis in various compartments of the adrenal gland and its potential influence in associated tumors, including pheochromocytomas and paragangliomas. Here, we provide an overview of the most recent findings on the hypoxia signaling pathway in vivo, including its role in the endocrine system, especially in adrenal tumors. [ABSTRACT FROM AUTHOR]

MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing's syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing's disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes. [ABSTRACT FROM AUTHOR]

The adrenal gland and its hormones regulate numerous fundamental biological processes; however, the impact of hypoxia signaling on adrenal function remains poorly understood. Here, we reveal that deficiency of HIF (hypoxia inducible factors) prolyl hydroxylase domain protein-2 (PHD2) in the adrenal medulla of mice results in HIF2α-mediated reduction in phenylethanolamine N-methyltransferase (PNMT) expression, and consequent reduction in epinephrine synthesis. Simultaneous loss of PHD2 in renal erythropoietin (EPO)-producing cells (REPCs) stimulated HIF2α-driven EPO overproduction, excessive RBC formation (erythrocytosis), and systemic hypoglycemia, which is necessary and sufficient to enhance exocytosis of epinephrine from the adrenal medulla. Based on these results, we propose that the PHD2-HIF2α axis in the adrenal medulla regulates the synthesis of epinephrine, whereas in REPCs, it indirectly induces the release of this hormone. Our findings are also highly relevant to the testing of small molecule PHD inhibitors in phase III clinical trials for patients with renal anemia. Key messages: HIF2α and not HIF1α modulates PNMT during epinephrine synthesis in chromaffin cells. The PHD2-HIF2α-EPO axis induces erythrocytosis and hypoglycemia. Reduced systemic glucose facilitates exocytosis of epinephrine from adrenal gland. [ABSTRACT FROM AUTHOR]

The hypothalamus-pituitary- adrenal (HPA) axis is activated in response to inflammation leading to increased production of anti-inflammatory glucocorticoids by the adrenal cortex, thereby representing an endogenous feedback loop. However, severe inflammation reduces the responsiveness of the adrenal gland to adrenocorticotropic hormone (ACTH), although the underlying mechanisms are poorly understood. Here, we show by transcriptomic, proteomic, and metabolomic analyses that LPS-induced systemic inflammation triggers profound metabolic changes in steroidogenic adrenocortical cells, including downregulation of the TCA cycle and oxidative phosphorylation, in mice. Inflammation disrupts the TCA cycle at the level of succinate dehydrogenase (SDH), leading to succinate accumulation and disturbed steroidogenesis. Mechanistically, IL-1β reduces SDHB expression through upregulation of DNA methyltransferase 1 (DNMT1) and methylation of the SDHB promoter. Consequently, increased succinate levels impair oxidative phosphorylation and ATP synthesis and enhance ROS production, leading to reduced steroidogenesis. Together, we demonstrate that the IL-1β- DNMT1-SDHB- succinate axis disrupts steroidogenesis. Our findings not only provide a mechanistic explanation for adrenal dysfunction in severe inflammation, but also offer a potential target for therapeutic intervention. [ABSTRACT FROM AUTHOR]