11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche

Abstract

Context: Adrenarche refers to the rise of dehydroepiandrosterone sulfate (DHEA-S) associated with the development of a functional adrenal zona reticularis. Clinical features of adrenarche include onset of body odor, axillary hair, and pubic hair, which reflect increased androgen action. An early rise in adrenal androgens, or premature adrenarche (PremA), is a risk factor for adverse metabolic profiles in adolescence and adulthood. The bioactive androgens associated with adrenarche and PremA remain poorly understood. The adrenal gland is a potential source of testosterone (T) and the 11-oxygenated derivatives 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT).

Objective: The objective of this study was to characterize the adrenal androgen biome contributing to adrenarche and PremA.

Participants and methods: With the use of mass spectrometry, 19 steroids including the 11-oxygenated derivatives of T were measured in sera obtained from girls with PremA (n = 37; 4 to 7 years) and age-matched girls (n = 83; 4 to 10 years).

Results: In reference population girls, dehydroepiandrosterone, DHEA-S, androstenediol-3-sulfate, T, and 11KT all increased at the onset of adrenarche (6 to 8 years) and beyond (9 to 10 years) (P < 0.05 vs younger subjects 4 to 5 years). T, 11OHT, and 11KT were further elevated in PremA vs age-matched girls (P < 0.001). Circulating concentrations of 11KT during adrenarche and PremA exceeded those of T and 11OHT (11KT > T ≥ 11OHT). Androgen receptor activity and nuclear translocation studies demonstrated that 11KT is a potent androgen similar to T.

Conclusions: Our findings suggest that 11KT is the dominant bioactive androgen in children during adrenarche and PremA. Its androgenic capacity suggests that it may be responsible for the phenotypic changes seen in these phenomena.

Figure 1.

Steroidogenic pathways depicting adrenal mineralocorticoid, glucocorticoid, and androgen biosyntheses. The steroidogenic enzymes and cofactor proteins are marked in blue. The 11-oxygenated androgens are highlighted in black. Steroids not quantified are marked in gray. Dashed arrow denotes minor conversion to product. 11KA, 11-ketoandrostenedione; 11OHA, 11β-hydroxyandrostenedione.

Figure 2.

Androgen studies using the CV1-ARLuc cell line. (A) Gaussia reporter gene regulation in response to unconjugated and sulfated C₁₉ steroids. The data represent the mean ± SEM of three independent experiments, each performed in triplicate. *P < 0.05 vs basal. Basal denotes treatment with 0.1% methanol (vehicle). (B) T and 11KT binding causes AR nuclear translocation. Fluorescence microscopy of CV1-ARLuc cells stably transfected with a human AR and treated with or without 100 nM of T and 11KT, respectively, for 18 hours. Green fluorescence represents AR immunoreactivity, and blue fluorescence is DAPI (nucleus). (C) The 50 most highly upregulated genes in CV1-ARLuc cells after treatment with 100 nM of T and 11KT as identified by RNA-Seq. Both T and 11KT stimulated 41 of these 50 genes. (D) RNA-Seq and (E) quantitative RT-PCR validation of the expression of CV1-ARLuc genes stimulated by treatment with T and 11KT. These genes were previously shown to be androgen responsive. The data represent the mean ± SEM of three independent experiments, each performed in triplicate. *P < 0.05, T vs 11KT. AD, androstenedione; DAPI, 4′,6-diamidino-2-phenylindole.