Multiple androgen pathways contribute to the steroid signature of adrenarche.

CONTEXT: Adrenarche is a normal developmental event in mid-childhood characterized by increasing adrenal androgen secretion. The role of the classic androgen pathway has been well described in adrenarche, but the role of newer active androgens and additional androgen pathways is less clear. OBJECTIVE: To study the contribution of novel androgens and related steroid biosynthesis pathways to the development of adrenarche, and to identify additional steroid biomarkers of adrenarche. DESIGN: A longitudinal study of children aged 6-8 years at baseline, followed up at ages 8-10 and 14-16 years. A total of 34 children (20 girls) with clinical and/or biochemical signs of adrenarche (cases) and 24 children (11 girls) without these signs (controls) at age 8-10 years were included. Serum steroid profiling was performed by liquid chromatography high-resolution mass spectrometry. MAIN OUTCOME MEASURES: Thirty-two steroids compartmentalized in progestagens, gluco- and mineralocorticoid pathways, and four androgen related pathways, including the classic, backdoor, 11-oxy, and 11-oxy backdoor pathways. RESULTS: The classic and 11-oxy androgen pathways were more active, and serum concentrations of main androgens in the classic (dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione and androsterone) and 11-oxy (11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, 11-ketoandrostenedione, and 11-ketotestosterone) pathways were higher in cases at ages 6-8 and 8-10 years. Pregnenolone concentrations at adrenarchal age (8-10 years) and cortisol concentrations at adolescence (14-16 years) were higher in cases. 11ß-hydroxyandrosterone and 11-ketoandrosterone tended to be higher in cases with clinical signs compared to cases who had only biochemical evidence of adrenarche, albeit they were detected at low levels. In biomarker analyses, calculated steroid ratios with cortisol, cortisone, or 11-deoxycortisone as dividers were better classifiers for adrenarche than single steroids. Among these ratios, androstenedione/cortisone was the best. CONCLUSIONS: The classic and 11-oxy androgen pathways are active in adrenarche. Children with earlier timing of adrenarche have higher serum cortisol levels at late pubertal age, suggesting that early adrenarche might have long-term effects on adrenal steroidogenesis by increasing the activity of the glucocorticoid pathway. Future studies should employ comprehensive steroid profiling to define novel classifiers and biomarkers for adrenarche and premature adrenarche.

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis.

Cholesterol is the precursor of all steroids, but how cholesterol flux is controlled in steroidogenic tissues is poorly understood. The cholesterol exporter ABCG1 is an essential component of the reverse cholesterol pathway and its global inactivation results in neutral lipid redistribution to tissue macrophages. The function of ABCG1 in steroidogenic tissues, however, has not been explored. To model this, we inactivated Abcg1 in the mouse adrenal cortex, which led to an adrenal-specific increase in transcripts involved in cholesterol uptake and de novo synthesis. Abcg1 inactivation did not affect adrenal cholesterol content, zonation, or serum lipid profile. Instead, we observed a moderate increase in corticosterone production that was not recapitulated by the inactivation of the functionally similar cholesterol exporter Abca1. Altogether, our data imply that Abcg1 controls cholesterol uptake and biosynthesis and regulates glucocorticoid production in the adrenal cortex, introducing the possibility that ABCG1 variants may account for physiological or subclinical variation in stress response.

Mitochondrial dysfunction results in enhanced adrenal androgen production in H295R cells.

The role of mitochondria in steroidogenesis is well established. However, the specific effects of mitochondrial dysfunction on androgen synthesis are not fully understood. In this study, we investigate the effects of various mitochondrial and metabolic inhibitors in H295R adrenal cells and perform a comprehensive analysis of steroid and metabolite profiling. We report that mitochondrial complex I inhibition by rotenone shifts cells toward anaerobic metabolism with a concomitant hyperandrogenic phenotype characterized by rapid stimulation of dehydroepiandrosterone (DHEA, 2 h) and slower accumulation of androstenedione and testosterone (24 h). Screening of metabolic inhibitors confirmed DHEA stimulation, which included mitochondrial complex III and mitochondrial pyruvate carrier inhibition. Metabolomic studies revealed truncated tricarboxylic acid cycle with an inverse correlation between citric acid and DHEA production as a common metabolic marker of hyperandrogenic inhibitors. The current study sheds light on a direct interplay between energy metabolism and androgen biosynthesis that could be further explored to identify novel molecular targets for efficient treatment of androgen excess disorders.

Late diagnosis of partial 3ß-hydroxysteroid dehydrogenase type 2 deficiency - characterization of a new genetic variant.

Summary: Congenital adrenal hyperplasia (CAH) is one of the most common inherited rare endocrine disorders. This case report presents two female siblings with delayed diagnosis of non-classical CAH 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2D/HSD3B2) despite early hospital admission and apparent CAH manifestations such as infections, hirsutism, menstrual disturbances, and PCOS phenotype. Initially, sister 1 was misdiagnosed with PCOS and then 11-hydroxylase deficiency (CYP11B1), based on ultrasound, biochemical findings, and negative genetic testing for 21-hydroxylase deficiency (CYP21A2). Additional diagnostic workup was performed when sister 2also presented with symptoms of androgen excess. Genetic testing for CAH/steroid disorders finally revealed that both siblings were compound heterozygous for two variants in the HSD3B2 gene: a frameshift variant, c.558dup, p.(Thr187Hisfs*17) and a novel missense variant, c.65T>C, p.(Leu22Ser). A Synacthen test showed an insufficient cortisol increase. In vitro studies of the variants in a cell model revealed loss of function for the p.(Thr187Hisfs*17) and partial activity for p.(Leu22Ser) confirming non-classic CAH. Overlapping symptomatology and lack of specialized knowledge on steroid biosynthesis and associated rarest forms of CAH may explain the delayed diagnosis. However, with newer diagnostic methods comprising a less biased approach, very rare forms of non-classical CAH may no longer be overlooked in the future. Learning points: Non-classic 3ßHSD2 is likely underdiagnosed. Late diagnosis of mild non-classic 3ßHSD2 does occur and one should be aware of this diagnosis. Early diagnosis of NCCAH may prevent many consequences such as severe hirsutism, prolonged menstrual irregularities, infertility, or even adrenal crisis with severe infections. Comprehensive steroid profiling and genetic testing should be used earlier, especially when in doubt about a diagnosis.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

OBJECTIVE: Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. DESIGN: Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. METHODS: On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. RESULTS: The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. CONCLUSION: Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Activation of endogenous glucocorticoids by HSD11B1 inhibits the antitumor immune response in renal cancer.

Although immune-based therapies have revolutionized the management of cancer, novel approaches are urgently needed to improve their outcome. We investigated the role of endogenous steroids in the resistance to cancer immunotherapy, as these have strong immunomodulatory functions. Using a publicly available database, we found that the intratumoral expression of 11 beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), which regenerates inactive glucocorticoids into active glucocorticoids, was associated with poor clinical outcome and correlated with immunosuppressive gene signatures in patients with renal cell carcinoma (RCC). HSD11B1 was mainly expressed in tumor-infiltrating immune myeloid cells as seen by immunohistochemistry in RCC patient samples. Using peripheral blood mononuclear cells from healthy donors or immune cells isolated from the tumor of RCC patients, we showed that the pharmacological inhibition of HSD11B1 improved the response to the immune checkpoint inhibitor anti-PD-1. In a subcutaneous mouse model of renal cancer, the combination of an HSD11B1 inhibitor with anti-PD-1 treatment increased the proportion of tumor-infiltrating dendritic cells. In an intrarenal mouse tumor model, HSD11B1 inhibition increased the survival of mice treated with anti-PD-1. In addition, inhibition of HSD11B1 sensitized renal tumors in mice to immunotherapy with resiquimod, a Toll-like receptor 7 agonist. Mechanistically, we demonstrated that HSD11B1 inhibition combined with resiquimod increased T cell-mediated cytotoxicity to tumor cells by stimulating the antigen-presenting capacity of dendritic cells. In conclusion, these results support the use of HSD11B1 inhibitors to improve the outcome of immunotherapy in renal cancer and highlight the role of the endogenous glucocorticoid metabolism in the efficacy of immunotherapy.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.