Endocrine Abstracts

Introduction: AKR1D1 is a 5β-reductase that sits at the interface of steroid hormone metabolism and primary bile acid biosynthesis. 5β-reduced androgens are widely believed to be inactive, but to our knowledge there are currently no data that have directly tested their ability to activate the androgen receptor (AR). We therefore wanted to test the ability of AKR1D1 to regulate androgen availability in vitro and to determine if 5β-reduced androgens are able to activate the AR.

Methods: Human embryonic kidney cells (HEK293), that do not express AKR1D1, were transfected with either empty vector (EV) or AKR1D1. Cells were treated with testosterone (T), 5α-dihydrotestosterone (5α-DHT), androstenedione (A4) or 11-ketotestosterone (11-KT) (all 200 nM) for 24 h. Media was collected for further analysis. Testosterone clearance was determined using a commercially available ELISA assay. The ability of androgens, their 5β-reduced metabolites, and the collected media from the AKR1D1 overexpression experiments, to activate the AR was determined using HEK293 cells co-transfected with AR and an ARE-luciferase construct.

Results: Successful AKR1D1 overexpression was confirmed using qPCR and Western Blot analysis. AKR1D1 over-expression increased T clearance (P < 0.001). Using the AR/ARE co-transfection system, T and 5α-DHT were able to activate the AR. In contrast, 5β-DHT was inactive. In experiments using the conditioned media, AKR1D1 overexpression significantly reduced the ability of T, A4 and 11KT to activate the androgen receptor (P < 0.003, P < 0.02 and P < 0.007, respectively).

Discussion and conclusion: We have demonstrated that 5β-reduced androgens are unable to activate the AR. In addition, we have shown that AKR1D1 clears androgens, including T, A4 and 11KT, and therefore has a potent role to regulate androgen availability and action. AKR1D1 is highly expressed in the liver, and it is dysregulated in diabetes and NAFLD. Alterations in androgen availability as a consequence of changes in AKR1D1 activity and expression, may be crucial in the pathogenesis of NAFLD.