The A-ring reduction of 11-ketotestosterone is efficiently catalysed by AKR1D1 and SRD5A2 but not SRD5A1

Article abstract of DOI:10.1016/j.jsbmb.2020.105724

Testosterone and its 5α-reduced form, 5α-dihydrotestosterone, were previously thought to represent the only active androgens in humans. However, recent studies have shown that the potent androgen, 11-ketotestosterone, derived from the adrenal androgen precursor, 11β-hydroxyandrostenedione, may in fact serve as the primary androgen in healthy women. Yet, despite recent renewed interest in these steroids, their downstream metabolism has remained undetermined. We therefore set out to investigate the metabolism of 11-ketotestosterone by characterising the 5α- or 5β-reduction commitment step. We show that inactivation of 11-ketotestosterone is predominantly driven by AKR1D1, which efficiently catalyses the 5β-reduction of 11-ketotestosterone, committing it to a metabolic pathway that terminates in 11-ketoetiocholanolone. We demonstrate that 5α-reduction of 11-ketotestosterone is catalysed by SRD5A2, but not SRD5A1, and terminates in 11-ketoandrosterone, but is only responsible for a minority of 11-ketotestosterone inactivation. However, as 11-ketoetiocholanolone is also generated by the metabolism of the glucocorticoid cortisone, 11-ketoandrosterone should be considered a more specific urinary marker of 11-ketotestosterone production.

Full text of DOI:10.1016/j.jsbmb.2020.105724

Journal Pre-proof
The A-ring reduction of 11-ketotestosterone is efficiently catalysed by
AKR1D1 and SRD5A2 but not SRD5A1
Lise Barnard, Nikolaos Nikolaou, Carla Louw, Lina Schiffer, Hylton
Gibson, Lorna C. Gilligan, Elena Gangitano, Jacky Snoep, Wiebke
Arlt, Jeremy W. Tomlinson, Karl-Heinz Storbeck
PII:
S0960-0760(20)30249-1
DOI:
https://doi.org/10.1016/j.jsbmb.2020.105724
SBMB 105724
Reference:
To appear in:
Journal of Steroid Biochemistry and Molecular Biology
Received Date:
Revised Date:
Accepted Date:
6 March 2020
25 June 2020
30 June 2020
Please cite this article as: Barnard L, Nikolaou N, Louw C, Schiffer L, Gibson H, Gilligan LC,
Gangitano E, Snoep J, Arlt W, Tomlinson JW, Storbeck K-Heinz, The A-ring reduction of
1
1-ketotestosterone is efficiently catalysed by AKR1D1 and SRD5A2 but not SRD5A1,
Journal of Steroid Biochemistry and Molecular Biology (2020),
doi: https://doi.org/10.1016/j.jsbmb.2020.105724
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The A-ring reduction of 11-ketotestosterone is efficiently catalysed by AKR1D1 and
SRD5A2 but not SRD5A1
a#
b#
a
c
a
c
Lise Barnard , Nikolaos Nikolaou , Carla Louw , Lina Schiffer , Hylton Gibson , Lorna C. Gilligan ,
b,d
a,e
c,f
b
a,c
Elena Gangitano , Jacky Snoep , Wiebke Arlt , Jeremy W. Tomlinson , Karl-Heinz Storbeck *
aDepartment of Biochemistry, Stellenbosch University, Stellenbosch, 7600, South Africa
bOxford Centre for Diabetes, Endocrinology and Metabolism, NIHR Oxford Biomedical Research
Centre, University of Oxford, Churchill Hospital, Oxford, OX3 7LE, UK
cInstitute of Metabolism and Systems Research, University of Birmingham, Birmingham, B15 2TT,
UK
d
Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy
e
Molecular Cell Physiology, VU - Amsterdam, The Netherlands.
fNIHR Birmingham Biomedical Research Centre, University of Birmingham and University Hospitals
Birmingham NHS Foundation Trust, Birmingham, B15 3GW, UK
#
Joint first authors
*Corresponding author
Highlights




SRD5A2, but not SRD5A1 catalyses the 5α-reduction of 11-ketotestosterone (11KT)
11-ketotestosterone is efficiently 5β-reduced by AKR1D1
5β-reduction of 11KT leads to the generation of 11-ketoetiocholanolone (11-KEt)
11-KEt is the major product of 11KT metabolism in human hepatoma cells
Abstract
Testosterone and its 5α-reduced form, 5α-dihydrotestosterone, were previously thought to
represent the only active androgens in humans. However, recent studies have shown that the
potent androgen, 11-ketotestosterone, derived from the adrenal androgen precursor,
11β-hydroxyandrostenedione, may in fact serve as the primary androgen in healthy women. Yet,
despite recent renewed interest in these steroids, their downstream metabolism has remained
undetermined. We therefore set out to investigate the metabolism of 11-ketotestosterone by
characterising the 5α- or 5β-reduction commitment step. We show that inactivation of
11-ketotestosterone is predominantly driven by AKR1D1, which efficiently catalyses the
1

5β-reduction of 11-ketotestosterone, committing it to a metabolic pathway that terminates in
11-ketoetiocholanolone. We demonstrate that 5α-reduction of 11-ketotestosterone is catalysed by
SRD5A2, but not SRD5A1, and terminates in 11-ketoandrosterone, but is only responsible for a
minority of 11-ketotestosterone inactivation. However, as 11-ketoetiocholanolone is also generated
by the metabolism of the glucocorticoid cortisone, 11-ketoandrosterone should be considered a
more specific urinary marker of 11-ketotestosterone production.
Abbreviations
11KA4,
11-ketoandrostenedione,
11KAn,
11-ketoandrosterone;
11K-5α-dione,
11K-5α-androstanedione; 11K-5α-DHT, 11-keto-5α-dihydrotestosterone; 11K-5β-dione, 11K-5β-
androstanedione; 11K-5β-DHT, 11-keto-5β-dihydrotestosterone; 11KEdiol, 11-ketoetiocholanediol;
1
1
1
1KEt,
11-ketoetiocholanolone;
11KT,
11-ketotestosterone,
11OHA4,
11OHEt,
1β-hydroxyandrostenedione;
11OHAn,
11β-hydroxyandrosterone;
1β-hydroxyetiocholanolone; 11OHT, 11β-hydroxytestosterone; HSD17B2, 17β-hydroxysteroid
dehydrogenase type 2; 5α-DHT, 5α-dihydrotestosterone; 5α-dione, 5α-androstanedione; 5β-DHT,
β-dihydrotestosterone; 5β-dione, 5β-androstanedione; A4, androstenedione; Adiol,
5
androstanediol; AKR1C3, 17β-hydroxysteroid dehydrogenase type 5; AKR1C4; 3α-hydroxysteroid
dehydrogenase type 1; AKR1D1, aldo-keto reductase family member 1 type D1; An, androsterone;
AR, androgen receptor; CYP11B1, cytochrome P450 11β-hydroxylase; Ediol, etiocholanediol; Et,
etiocholanolone; SRD5A1, steroid 5α-reductase type 1; SRD5A2, steroid 5α-reductase type 2; T,
testosterone; UHPSFC-MS/MS, ultra-high performance supercritical fluid chromatography tandem
mass spectrometry.
Keywords
11-oxygenated androgens; 11-ketotestosterone; steroid 5β-reductase; steroid 5α-reductase;
11-ketoetiocholanolone; steroid metabolism
1. Introduction
Although once forgotten, several recent studies have shown that 11-oxygenated androgens
play an important role in human androgen biology [1–3]. Their precursor,
1β-hydroxyandrostenedione (11OHA4), is an abundant product of adrenal steroidogenesis [4]
produced by the 11β-hydroxylation of androstenedione (A4), catalysed by the cytochrome P450
1
11β-hydroxylase (CYP11B1) [5]. 11OHA4 can subsequently be converted to
11-ketoandrostenedione (11KA4) in peripheral tissues expressing 11β-hydroxysteroid
dehydrogenase type 2 (HSD11B2) [5,6]. 11KA4 in turn serves as a preferred substrate for the
androgen-activating enzyme 17β-hydroxysteroid dehydrogenase type 5 (HSD17B5, also known as
AKR1C3) [6,7], yielding 11-ketotestosterone (11KT), which binds to and activates the human
androgen receptor (AR) with an affinity and potency comparable to that of testosterone (T) [6,8].
2

Several studies have confirmed that the aforementioned 11-oxygenated androgens are present in
circulation for both men and women [4,9–14]. Significantly, the circulating concentrations of 11KT
have been shown to be equal to or higher than that of T in healthy women [11,13,14], and unlike
the levels of T, appear not to decrease following menopause [13] or fluctuate during the menstrual
cycle [14]. Moreover, significantly increased serum concentrations of 11KT and its precursors have
been measured in patients with polycystic ovary syndrome and congenital adrenal hyperplasia, with
11KT being suggested to be the primary active androgen in these conditions [10,11,15].
Given the abundance and clear physiological importance of 11KT, it is unusual that this steroid
went undetected in humans for so long. Failure to detect 11KT in circulation is likely explained by
the predominant use of immunoassays, which focus on measuring single metabolites such as T,
and often use antibodies that do not distinguish between closely related steroid metabolites [16].
While 11β-hydroxyandrosterone (11OHAn) has been described as a urinary steroid metabolite
reflecting the 11-oxygenated androgen pathway using gas chromatography-mass spectrometry
(
GC-MS) analysis [17,18], this metabolite is mostly derived from 11OHA4, with direct downstream
metabolites of 11KT not yet described [19,20]. This led us to hypothesise that the urinary
metabolite(s) of 11KT may be masked by other predominant urinary metabolites. In order to
investigate this, we considered the standard metabolic pathways for the metabolism of T, which
4
contains the same 3-keto-∆ -steroid moiety as 11KT [20].
The first step in T metabolism is either the 5α- or the 5β-reduction of carbon 5, both
physiologically irreversible reactions that commit T to metabolism and excretion (Figure 1) [20–23].
5
α-Reduction is catalysed by the 5α-reductase isozymes, SRD5A1 and SRD5A2 [22], which are
expressed in a tissue specific manner [24,25]. While the resulting product, 5α-dihydrotestosterone
5α-DHT) is a more potent androgen than T, further metabolism yields inactive metabolites [26–
8].
(
2
5
β-Reduction is catalysed by a single enzyme, aldo-keto reductase family 1 member D1
AKR1D1; also known as 5β-steroid reductase) [21,29]. AKR1D1 is essential to both bile acid
biosynthesis and steroid hormone inactivation and clearance [30,31]. The AKR1D1-catalysed
β-reduction of T yields 5β-dihydroxytestosterone (5β-DHT), a metabolite that has no androgenic
(
5
activity due to the induction of a conformational change in the A/B-ring that hinders binding to the
AR [32,33]. AKR1D1 is primarily expressed in the liver [34–37] were it competes with SRD5A
4
isoforms for 3-keto-Δ steroid substrates [21,24]. The relative expression levels and activity of these
enzymes will therefore determine the ratio of 5α- and 5β-reduced products.
Both 5α-DHT and 5β-DHT are subsequently 3α-reduced by 3α-hydroxysteroid dehydrogenase
type 3/1 (AKR1C2/4) yielding androstanediol (Adiol) and etiocholanediol (Ediol), respectively
[
38,39]. These metabolites are subject to metabolism by oxidative 17β-hydroxysteroid
dehydrogenases (HSD17B) yielding androsterone (An) and etiocholanolone (Et), respectively, the
primary T metabolites measured in urine [20]. Applying the same principles to the metabolism of
11KT would yield the end-products, 11-ketoandrosterone (11KAn) and 11-ketoetiocholanolone
3

(
11KEt) (Figure 1). Although we have previously demonstrated the 5α-reduction of 11KT by
SRD5A1 and SRD5A2 [6], these reactions could not be quantified due to a lack of reference
material at the time. Lower circulating concentrations of 11-keto-5α-dihydrotestosterone
(
11K-5α-DHT) than 5α-DHT suggest that 11KT is not as efficiently 5α-reduced as T [40].
Conversely, the AKR1D1 catalysed 5β-reduction of 11KT has not been investigated to date. The
proposed downstream metabolite, 11KEt, is routinely measured in urine as a prominent urinary
steroid metabolite derived from cortisone [41–48].
In this study, we characterised AKR1D1, SRD5A1 and SRD5A2 activity towards T and 11KT
using progress curve analysis and computational modelling. We demonstrate for the first time that
the sequential metabolism of 11KT by AKR1D1, AKR1C2 and 17β-hydroxysteroid dehydrogenase
type 2 (HSD17B2) yields the steroid 11KEt, which is likely the primary urinary metabolite of 11KT.
2. Materials and methods
2.1 Cell lines
HEK293 and HepG2 cells were purchased from America Type Culture Collection and were
validated by STR profile analysis. Huh7 were purchased from the Japanese Cancer Research
Resources Bank, where the cells were validated by isozyme analysis. All experiments were
conducted within 20-passage numbers from purchase or revalidation. HEK293 cells were cultured
in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS),
100 units/mL of penicillin and 100 µg/mL streptomycin. HepG2 and Huh7 cells were cultured in
DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 4.5 g/L glucose and 1% non-
essential amino acids (Thermo Fisher Scientific)). Culturing conditions were kept stable at 37˚C,
9
0% relative humidity and 5% CO
using MycoAlertTM mycoplasma detection kit (LT07-318) or MycoAlertTM assay control set
LT07-518) from Lonza.
2
. All cells were regularly tested for mycoplasma contamination
(
2.2 Plasmid constructs
The pCMV6-XL4/AKR1D1 plasmid construct (CAT#: SC116410) was purchased from
Origene Technologies. The pCMV7/SRD5A1 and pCMV7/SRD5A2 plasmid constructs were gifts
from Prof. D.W. Russell (UT Southwestern Medical Centre, Dallas, USA). The pcDNA3/AKR1C2
and pcDNA4/17βHSD2 plasmid constructs were gifts from Prof. T.M. Penning (University of
Pennsylvania, School of Medicine, Philadelphia, USA) and Prof. J. Adamski (Institute of
Experimental Genetics, Helmholtz Zentrum München, Germany), respectively. Plasmid inserts
were confirmed by sequence analysis.
2.3 Steroids
4

1
1β-hydroxyandrostendione (11OHA4), 11β-hydroxytestosterone (11OHT), 11-Keto-5α-
dihydrotestosterone (11K-5α-DHT), 11-ketoandrosterone (11KAn), 11-ketoetiocholanolone
11KEt), 11-ketotestosterone (11KT), androsterone (An) and testosterone (T) were purchased from
(
Steraloids. Deuterated 11β-hydroxyandrostenedione 2,2,4,6,6,16,16-D7 (D7-11OHA4) 98%,
androstenedione 2,2,4,6,6,16,16-D7 (D7-A4) 98%, cortisol 9,11,12,12-D4 (D4-F) 98% and
testosterone 1,2-D2 (D2-T) 98% were obtained from Cambridge Isotopes. Testosterone 16,16,17-
D3 (D3-T) and 5α-dihydrotestosterone 16,16,17-D3 (D3-DHT) solutions from Cerilliant® were
purchased from Sigma-Aldrich, while 11-ketotestosterone16,16,17-D3 (D3-11KT) and
androsterone 2,2,4,4-D4 (D4-An) solutions were obtained from IsoSciences.
5

2.4 Enzyme assays in transiently transfected HEK293 cells
5
HEK293 cells were seeded into 100 mm dishes at a cell density of 2x10 cells/mL in a total
volume of 10 mL. Following a 24-hour incubation, each dish was transfected with 10 µg of either
pCMV6-XL4/AKR1D1, pCMV6/SRD5A1, pCMV6/SRD5A2 or pCIneo (as negative control) using
XtremeGene HP® DNA transfection reagent (Roche) according to the supplier’s protocol. After a
further 24 hours, cells were replated into 48-well Corning® CELLBIND® plates at a cell
5
concentration of 1x10 cells/mL in 250 µL. For co-expression investigations, AKR1D1 and either
5
SRD5A1 or SRD5A2 transfected cells were mixed in a 1:1 ratio and 500 µL (1x10 cell/mL) plated
into 24-well Corning® CELLBIND® plates. Cells were incubated for a further 24 hours after which
the media was replaced with media containing the appropriate steroid substrates. These included
0
.1, 1 and 10 µM A4, T, 11KA4 or 11KT. Aliquots of 250 µL were collected from 48-well plates at
, 2, 3, 4, 6, 8, 10 and 12 hours. A4 and 11KA4, and T and 11KT samples were combined allowing
1
for multiplexing during ultra-high performance supercritical fluid chromatography tandem mass
spectrometry (UHPSFC-MS/MS) analysis. Aliquots of 500 µL were collected after 24 hours from
24-well plates containing SRD5A1/2 and AKR1D1 co-expressing cells.
2.5 Promoter reporter assays
To determine AR activation, HEK293 cells were transiently transfected with either pCMV6-
XL4/AKR1D1 or empty plasmid vector for 48 hours as previously described [49], followed by
treatments with unsupplemented phenol-red free DMEM (Thermo Fisher Scientific) containing 200
nM of either T or 11KT for 24 hours. Cell media aliquots (500 µL) were collected and stored at -
2
0oC. In another set of experiments, HEK293 cells were transiently co-transfected with a
pcDNA3.1+AR construct and an androgen responsive element (ARE) reporter - a mixture of an
inducible ARE-responsive firefly luciferase construct and a constitutively expressing renilla
luciferase construct (#CCS-1019L, QIAgen). After 48 hours, cell media was replaced with the stored
steroid containing media aliquots described above, and cells were incubated for 24 hours. Cell
lysates were then harvested in passive lysis buffer, and reporter activity was measured using the
Luciferase Assay System (Promega) and an EnSpire Multimode plater reader (PerkinElmer). The
data were presented as the ratio of firefly to renilla luciferase activity (Fluc/Rluc) in percentage.
2.6 Sequential metabolism of 11KT using conditioned media
AKR1D1, AKR1C2 and HSD17B2 transfections, performed as described above, were
5
staggered sequentially by 24 hours. First AKR1D1 transfected cells (2x10 cells/mL, 2 mL,
6-well plates) were incubated for 24 hours after which the media was removed and replaced with 2
mL media containing 4 µM T or 11KT. Following 24 hours, 1 mL conditioned media was carried
5
over to cells transfected with AKR1C2 (2x10 cells/mL, 2 mL, 6-well plate). The conditioned media
was diluted 1:1 with 1 mL fresh media. After a further 24 hours, 500 µL conditioned media was
6

5
transferred to cells transfected with HSD17B2 (2x10 cells/mL, 500 µL, 24-well plate) and incubated
a further 24 hours. A final steroid concentration of 2 µM was anticipated following the dilution of the
conditioned media. Aliquots (500 µL) were collected at each step for UPHSFC-MS/MS analysis.
2.7 Steroid extraction
All steroid samples were brought to room temperature before the addition of internal
standards (1.5 ng D2-T and 15 ng of D7-11OHA4, D7-A4 and D4-F for the HEK293 experiments or
0 ng D3-T, D3-11KT, D3-DHT, D4-An and D4-F for the HepG2 and Huh7 experiments). Steroids
1
were extracted using three volumes of tert-Butyl methyl ether (MTBE). Samples were mixed by
vortexing and incubated at -80°C for 1 hour. The organic layer was subsequently transferred to a
clean test tube and dried under a stream of nitrogen at 45°C. The dried steroid residue was
suspended in 50% MeOH in water (150 µL) and stored at -20°C until analysis by UHPSFC-MS/MS.
2.8 UHPSFC-MS/MS analyses of steroid metabolites
2
Steroid metabolites were separated and quantified using an ACQUITY UPC (Waters
Corporation) coupled to a Xevo-TQS (Waters Corporation) triple quadrupole mass spectrometer.
2
Chromatographic separation was achieved using an ACQUITY UPC Torus 1-AA column (3.0 mm
x 50 mm; 1.7 µm) (Waters Corporation) at 60°C with liquid CO (Solvent A) modified with absolute
2
MeOH (solvent B) as the mobile phases. A linear gradient increased solvent B from 20% to 30%
over 2.5 minutes at a flow rate of 1.2 mL/minute. The automated backpressure regulator was set
to 2000 psi. The make-up solvent consisted of 1% formic acid in MeOH and fed into the system at
a flow rate of 0.2 mL/minute. The injection volume was 2 µL. Steroids were quantified by multiple
reaction monitoring (MRM) in positive electrospray ionisation mode. The instrument settings were
as follows: source temperature: 150°C; capillary voltage: 3.8 kV; cone voltage: 25-125 V; cone gas
flow: 150 L/hour; desolvation temperature: 500°C; desolvation gas flow: 750 L/hour; collision gas
flow: 0.2 mL/minute. All data were collected and analysed using the MassLynx Software Package
(
Version 4.1) (Waters Corporation).
2.9 Determination of kinetic parameters for AKR1D1, SRD5A1 and SRD5A2 and computational
model construction
The time course data collected from HEK293 cells transiently transfected with SRD5A1,
SRD5A2 or AKR1D1 were used to determine kinetic parameters (apparent Vmax and K ) in Wolfram
m
Mathematica (version 12). Irreversible Michaelis-Menten enzyme kinetics were assumed for the
determination of all parameter values. An ordinary differential equation (ODE) based model was
constructed and fitted to the data. Non-linear regression was used to minimise the objective
function, which is the sum squared residual values (SSR) between the model fit and the data
weighted by the variance. The kinetic parameters were validated by their ability to predict the
7

experimental data from independent time course experiments. Differences in transfection
efficiencies between experiments were accounted for by measuring the initial rates of selected
substrates for each experiment.
The validated kinetic constants were subsequently used to construct a model of SRD5A1,
SRD5A2 and AKR1D1 co-expression. The model was validated by the ability to predict the
metabolism of T and 11KT by a 1:1 mixture of cells expressing SRD5A1 and AKR1D1 or SRD5A2
and AKR1D1. Following validation, the model was used to predict the metabolism of T or 11KT by
various combinations of SRD5A1, SRD5A2 and AKR1D1 activity. The computational model will be
made available on JWS online (https://jjj.bio.vu.nl/) upon acceptance of the manuscript, and is
available in SBML format upon request.
2.10 Steroid metabolism in Huh7 and HepG2 liver cell lines
HepG2 and Huh7 cells were plated into 24-well Corning® CELLBIND® plates at a cell
5
density of 1.6x10 cells/mL (500 µL/well). After 24 hours, cells were treated with either 20 nmol of
AKR1D1 siRNA or scrambled control (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen)
prepared in OPTIMEM serum free media (Invitrogen) as previously reported [49]. The cells were
incubated for 48 hours after which the media was removed and replaced with unsupplemented
phenol-red free DMEM (Thermo Fisher Scientific) containing 1 µM T or 11KT (500 µL/well). Cell
media samples were collected after 24 hours for Huh7 cells and 48 hours for HepG2 cells and
stored at -20˚C prior to steroid extraction.
2
.11 GC-MS analysis
Measurement of steroid metabolites was carried out by GC-MS as previously described
[
19,50]. In brief, free and conjugated steroids were extracted from 1 mL cell culture media by solid-
phase extraction. Steroid conjugates were enzymatically hydrolysed, re-extracted, and chemically
derivatised to form methyloxime trimethyl silyl ethers. GC-MS was performed on an Agilent 5975
instrument operating in selected-ion-monitoring (SIM) mode to achieve sensitive and specific
detection and quantification of 38 selected steroid metabolites.
2.12 Human samples
Human liver biopsies were collected from healthy female individuals subject to their written
informed consent (University of Oxford, Translational gastroenterology biobank ethics NRES ref:
6/YH/0247). Intraoperatively, a wedge biopsy was taken from the anatomical left lobe of the liver
1
after pneumoperitoneum was established. Liver tissue was washed in 0.9% NaCl and then stored
in RNA later stabilization solution at -80°C until RNA extraction.
8

2.13 RNA extraction and gene expression analysis
Total RNA was extracted from cells using Tri-Reagent (Sigma-Aldrich) and RNA
concentrations were determined spectrophotometrically (OD260
)
using
a
NanoDrop
spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed in a total
volume of 20 μL:1 μg of total RNA, incubated with 10x RT Buffer, 100 mM dNTP mix, 10x RT
Random Primers, 50 U/μL MultiScribe Reverse Transcriptase and 20 U/μL RNase Inhibitor (Thermo
Fisher Scientific). Reactions were performed under the following conditions: 25°C for 10 minutes,
37°C for 120 minutes and then terminated by heating to 85°C for 5 minutes.
All quantitative PCR (qPCR) experiments were conducted using a QuantStudio 7 Realtime
PCR System (Applied Biosystems). Reactions (6 μL) were performed in 384-well plates in reaction
buffer containing 3 μL of 2x Kapa Probe Fast qPCR Master Mix (Sigma-Aldrich). All probes were
supplied by Thermo Fisher Scientific as predesigned TaqMan Gene Expression Assays (FAM dye-
labelled). The reaction conditions were: 95°C for 3 minutes, then 40 cycles of 95°C for 3 seconds
and 60°C for 20 seconds. Relative expression of AKR1D1 in each sample was determined by a
pre-existing equation and expression values normalised to 18SrRNA, unless stated otherwise
[
37,51].
2.14 Cell lysis and immunoblotting
®
Cells lysis was performed using RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5%
sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) (Sigma-Aldrich), and protease inhibitor
cocktail (Thermo Fisher Scientific). Protein concentrations were measured using a commercially
available assay (Bio-Rad Laboratories Inc.) according to the manufacturer’s protocol. Primary
human AKR1D1 (dilution 1:500 - HPA057002, Atlas Antibodies AB), β-tubulin (#15115,
monoclonal) (Cell Signalling), and secondary antibodies (P044801-2, polyclonal) from Dako
(
Agilent) were used at a dilution of 1:1000 (primary) and 1:2000 (secondary) respectively. Bands
were visualised with Bio-Rad Clarity Western ECL and ChemiDocXS imager (Bio-Rad). Western
blots were quantified by densitometry analysis using ImageJ (NIH: http://rsb.info.nih.gov/ij),
normalised to β-tubulin to correct for variability in gel loading.
9

3. Results
3.1 11KT is efficiently metabolised by AKR1D1 and SRD5A2, but not SRD5A1
In order to evaluate the potential metabolism of 11KT, we characterised the activity of
SRD5A1, SRD5A2 and AKR1D1 towards T and 11KT using progress curve analyses (Figure 2 and
supplementary data: Figure 1). Both SRD5A2 and AKR1D1 catalysed the conversion of T and 11KT
to their respective 5α- and 5β-products. T was also effectively 5α-reduced by SRD5A1, however,
this isozyme did not efficiently catalyse the 5α-reduction of 11KT. While the determined apparent
K
m
values for T and 11KT were similar (0.55 µM vs 0.64 µM) for SRD5A1, the apparent Vmax was
0-fold higher for T compared to 11KT. The resulting ratio of Vmax/K , which serves as an estimate
of catalytic efficiency, therefore demonstrated the clear preference of SRD5A1 for T over 11KT
1
m
-1
-1
(
0.12 h vs 0.01 h ) (Table 1). Unlike SRD5A1, SRD5A2 efficiently catalysed the 5α-reduction of
both T and 11KT, although a preference for T was observed in both the apparent K
values (Table 1). No steroid conversion was observed in cells transfected with empty vector.
m
and Vmax/K
m
Notably, we show for the first time that 11KT is a substrate for AKR1D1. In fact, the Vmax
measured for 11KT was 16-fold greater than the Vmax for T (0.37 µM/h vs 0.02 µM/h). However, this
was offset by a lower apparent K
more efficient reaction for T as estimated from the Vmax/K
values for all three enzymes indicates that the order of estimated enzyme efficiency is
m
for T when compared to 11KT (Table 1), resulting in a moderately
-1
-1
m
values (0.24 h vs 0.09 h ). Comparing
the Vmax/K
m
SRD5A2>AKR1D1>SRD5A1 for both T and 11KT, but with significant differences observed
between the two substrates. A similar trend was observed for the androgen precursors A4 and
11KA4 (Supplementary data: Figure 1 and Table 1). Neither 11OHA4 nor 11β-hydroxytestosterone
(
11OHT) were metabolised by AKR1D1 (data not shown).
3.2 AKR1D1 catalyses the inactivation of 11KT
The 5α-reduction of T and 11KT yields 5α-DHT and 11K-5α-DHT, respectively, which are
both more potent agonists of the AR than their direct precursors [6,8]. Conversely, AKR1D1
catalysed 5β-reduction of T yields 5β-DHT, which unlike its precursor, is not an agonist of the AR
[
52]. Here we show that similar to the case with T, AKR1D1 inactivates 11KT (Figure 3),
demonstrating that 11K-5β-DHT is an inactive metabolite.
3.3 Co-expression of SRD5A and AKR1D1 favours the 5β-reduction of 11KT
Given that SRD5A1, SRD5A2 and AKR1D1 are co-expressed in the liver (Supplementary
data: Figure 3), the primary site of steroid metabolism, we next determined the effect of co-
expressing either SRD5A1 or SRD5A2 with AKR1D1. This was accomplished by transiently
transfecting HEK293 cells with either SRD5A1, SRD5A2 or AKR1D1 and then mixing SRD5A and
AKR1D1 transfected cells at a ratio of 1:1. The metabolism of T yielded 5α-DHT and 5β-DHT in
both cases, but with the 5α-reduced product dominating in the presence of SRD5A2 (Figure 4A and
1
0

C). Similarly, co-expression of SRD5A2 and AKR1D1 yielded more 11K-5α-DHT than 11K-5β-DHT
from incubations with 11KT, however the ratio of 5β:5α-products was 3.7-fold higher than that
observed for T (0.37 vs 0.10). Furthermore, co-expression of SRD5A1 and AKR1D1 yielded
minimal 5α-reduced product as expected due to the inability of SRD5A1 to efficiently catalyse the
5α-reduction of 11KT (Figure 3 and 4B, Table 1).
Metabolism of A4 and 11KA4 yielded similar trends to that of T and 11KT, with A4
metabolism leading to predominantly 5α-reduced products irrespective of which SRD5A isozyme
was co-expressed with AKR1D1 (Supplementary data: Figure 2A and C). Conversely, the co-
expression of SRD5A1 or SRD5A2 with AKR1D1 favoured the production of 11-keto-5β-
androstanedione (11K-5β-dione) or 11-keto-5α-androstanedione (11K-5α-dione), respectively
(
Supplementary data: Figure 2B and D). Neither 11OHA4 nor 11OHT showed significant
conversion to 5β-products in either co-expression system (data not shown).
Using the kinetic parameters established in Table 1, we constructed a computational model
for SRD5A and AKR1D1 co-expression. By adjusting the model for the activity of SRD5A and
AKR1D1 measured in the co-expression experiments, the model was able to accurately predict the
metabolism of T and 11KT in our experimental co-expression systems (Figure 4). Given the
accuracy of the model, we next used it as a tool to investigate the simultaneous expression of all
three enzymes. During these simulations we fixed the activity of SRD5A1 while varying the activity
of AKR1D1 and SRD5A2 10-fold in either direction. Using this approach we were able to show that
the proportion of 5α-reduced products remains lower for 11KT than for T under all simulated
conditions (Figure 5).
Model predictions for A4 and 11KA4 metabolism with the different levels of enzyme activity
were also generated (Supplementary data: Figure: 4). Similar results were observed with the
proportion of 5α-reduced products remaining lower for 11KA4 than for A4 under all simulated
conditions.
3.4 The sequential metabolism of 11KT by AKR1D1, AKR1C2 and HSD17B2 yields
11-ketoetiocholanolone
After showing that 11KT is efficiently 5β-reduced by AKR1D1, and that this 5β-reduction
makes a considerable contribution to 11KT metabolism under physiological conditions, we next
investigated the sequential metabolism of 11KT by AKR1D1, AKR1C2 and HSD17B2 in an effort
to replicate the established 5β-metabolic pathway followed by T (Figure 1). T was used as a control
in these experiments to ensure that the experimental system was able to produce Et (1 µM) from
T, thereby confirming the predicted enzymatic activities (data not shown). Using this system, we
demonstrated that 11KT is sequentially converted to 11KEt (1.34 µM) as anticipated (Figure 6).
Moreover, using UHPSFC-MS/MS we confirmed the formation of the predicted intermediate
products, 11K-5β-DHT and 11-keto-etiocholanediol (11KEdiol), though we were unable to quantify
these due to a lack of commercially available standards.
1
1

3.5 11KEt is the principal metabolite from 11KT metabolism in HepG2 cells
We next investigated the metabolism of 11KT and T in HepG2 human liver cells, which
endogenously express SRD5A1, AKR1D1, AKR1C2 and HSD17B2, but not SRD5A2 [28,37,53].
Using this model system, we were able to demonstrate the conversion of T to An and Et and 11KT
to 11KAn and 11KEt as predicted (Figure 7). The amount of 5β-reduced products were >10-fold
higher than that of the 5α-reduced products for both T and 11KT. The ratio of 5β:5α-products were,
however, higher for 11KT than T (22.7 vs 13.6) as predicted by our computational model (Figure
5).
To ensure that this observation was due to AKR1D1 we knocked down the expression of
AKR1D1 using siRNA (Figure 7A and B). This resulted in a significant reduction in the levels of both
Et and 11KEt. Furthermore, the ratios of 5β:5α products were significantly reduced for both T (13.6
to 2) and 11KT (22.7 to 4.7) (Figure 7E and H). Similar results were observed in Huh7 cells treated
with 11KT (Supplementary data: Figure 5).
4. Discussion
Although underappreciated for decades, it is now clear that 11-oxygenated androgens are
important components of the androgen pool in humans. Yet, despite recent renewed interest in
these steroids, the downstream metabolism of the potent androgen, 11KT, has remained
undetermined. Therefore, we set out to characterise the activity of AKR1D1 and SRD5A isozymes
4
towards T and 11KT, which accounts for the first commitment step of 3-keto Δ steroid metabolism
[
20]. Our results show for the first time that 11KT is a substrate for 5β-reduction by AKR1D1
Figure 3B, 4B and D and 5; Table 1), and that the resulting product 11K-5β-DHT cannot activate
(
the human AR (Figure 3). Moreover, we show that the 5α-reduction of 11KT is isozyme specific
with SRD5A2, but not SRD5A1, efficiently catalysing this reaction (Figure 3B, 4B and D; Table 1).
Considering that the liver is the primary site of steroid metabolism, and that both 5β- and
5α-reductases are expressed in this tissue (Supplementary data: Figure 3), we considered the
effect of the co-expression of SRD5A1, SRD5A2 and AKR1D1 using both experimental and
computational models. Our data demonstrates that the relative levels of AKR1D1 and SRD5A2
activity will regulate the metabolism of 11KT due to the limited SRD5A1 activity towards 11KT.
Higher ratios of AKR1D1:SRD5A2, as we report here for female liver (Supplementary data: Figure
3), will favour the flux towards the 5β-reduced metabolite, 11KEt (Figure 5.)
Up until now, 11KEt was believed to be an exclusive urinary metabolite of the glucocorticoid,
cortisone. Our data show that 11KT also contributes to 11KEt production, although its contribution
is likely lower due to the higher circulating levels of cortisone relative to 11KT [20]. Nonetheless,
this observation confirms that a major metabolite of 11KT is masked in urine metabolome assays
1
2

and explains why urinary metabolites have not been assigned to 11KT. This overlap also shows
that urinary 11KEt cannot be used as a surrogate to serum 11KT. Since we have illustrated that
11KA4 is also readily 5β-reduced by AKR1D1, with a 4-fold higher catalytic efficiency than 11KT
(
Table 1 and Supplementary data: Table 1), 11KA4 metabolism will also contribute to the production
of 11KEt. Our data also shows that a proportion of 11KT metabolism would yield 11KAn, which is
currently not routinely included in GC-MS urine analyses [19,54]. Furthermore, the metabolism of
11KA4, the direct precursor to 11KT, will also contribute to the production of 11KAn as 11KA4 is
efficiently 5α-reduced by both SRD5A1 and SRD5A2 (Table 1 and Supplementary data: Table 1).
Moreover, the glucocorticoid, cortisone, cannot be 5α-reduced and as a result cannot contribute to
the production of 11KAn. We therefore recommend that 11KAn is included as a urinary analyte in
future studies, since this serves as a specific marker for 11KT and its precursor, 11KA4, and is the
only marker of 11-oxygenated androgens not affected by glucocorticoid metabolism. Our data also
demonstrate that neither 11OHA4 nor 11OHT are substrates for AKR1D1 and as a result, their
further downstream metabolism would only contribute to urinary 11OHAn, and not to
11β-hydroxyetiocholanolone (11OHEt). 11OHAn is already generally accepted as a urinary marker
for 11OHA4, with minor (<10%) contribution from the glucocorticoid pathway [17,18].
Another significant finding from this study is that 11KT is not efficiently 5α-reduced by
SRD5A1, which is the predominant SRD5A isozyme expressed in the majority of peripheral tissues
[
22,24,25]. This observation likely explains why the circulating levels of 11K-5α-DHT are
substantially lower than those of 5α-DHT [40] and suggests that 11KT is the primary active 11-
oxygenated androgen. Moreover, 11KT may be resistant to peripheral inactivation by
glucuronidation [8,55]. Thus, it appears that the peripheral metabolism and inactivation of 11KT is
substantially different to that of T. We therefore propose that AKR1D1-catalysed 5β-reduction of
11KT plays a vital role in the inactivation and clearance of this potent androgen. When considering
the 11-oxygenated androgens tested in this study, it is interesting to note that AKR1D1
demonstrated a significant substrate preference towards the 11-keto substrates, 11KT and 11KA4,
and was unable to catalyse the 5β-reduction of the 11-hydroxysteroids, 11OHT or 11OHA4. We
have previously reported a similar finding with the androgen activating enzyme AKR1C3, which
efficiently catalyses the conversion of 11KA4 to 11KT, but does not accept 11OHA4 as a substrate
[
7]. Similarly, cortisone, which contains an 11-keto moiety, is preferentially 5β-reduced [34,56,57].
5. Conclusion
This study has shown for the first time that the AKR1D1-catalysed inactivation of 11KT leads
to the production of 11KEt, a urinary metabolite previously only associated with the metabolism of
the glucocorticoid cortisone. Furthermore, we have shown that 11KT is not a substrate for SRD5A1
and that it can only be efficiently 5α-reduced by SRD5A2. The relative expression ratios of
SRD5A2:AKR1D1 therefore determines the metabolic fate of 11KT, with higher hepatic levels of
1
3

AKR1D1 favouring the 5β-reduction of 11KT. Some 11KT is however 5α-reduced leading to the
production of 11KAn, which we propose should be used as a specific urinary marker for 11KT and
11KA4, as unlike 11KEt, it does not overlap with glucocorticoid metabolism.
Author Contributions
KHS conceived the original idea and designed the study. LB, NN, CL, HG LCG and EG planned and carried out
experiments and analyzed results, supervised by KHS, JWT, WA, and JS. LS and NN wrote the manuscript,
with support from KHS, JWT, WA and JS. All authors discussed the results and contributed to the drafting
and approval of the final manuscript
Acknowledgements
This work was supported by the Wellcome Trust (Investigator Grant WT209492/Z/17/Z, to
W.A.), the Academy of Medical Sciences UK (Newton Advanced Fellowship NAF004\1002, to
K.-H.S), the National Research Foundation (NRF) of South Africa (111622, to L.B), SARChI
DST/NRF grant (82813, to J.S), the European Society of Endocrinology (ESE) (Short-Term
Fellowship to E.G), the Medical Research Council, UK (programme grant to J.W.T. MR/P011462/1),
and the NIHR Oxford Biomedical Research Centre (J.W.T.). W.A. receives support from the
National Institute for Health Research (NIHR) Birmingham Biomedical Research Centre at the
University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham (Grant
Reference Number BRC-1215-20009). The views expressed are those of the author(s) and not
necessarily those of the NIHR UK, the Department of Health and Social Care UK or the NRF of
South Africa.
1
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2
0

Figure 1. Metabolism of T, 11KT and cortisone. Testosterone is either 5α-reduced or
β-reduced, followed by sequential 3α-reduction and 17β-oxidation reactions yielding the 5α-
5
product androsterone (An) and the 5β-product etiocholanolone (Et). The 5β-reduction of 11KT leads
to the production of 11-ketoetiocholanolone (11KEt), a metabolite also produced from the
metabolism of the glucocorticoid, cortisone.
Figure 2. Metabolism of 100 nM T (A) and 11KT (B) by AKR1D1, SRD5A1 and SRD5A2
expressed in non-steroidogenic HEK293 cells. Experimental results are shown as the mean
±
SD from a representative experiment used to determine the kinetic parameters. Model fits are
shown by the solid lines. Kinetic parameters were validated by their ability to predict the
experimental data from subsequent independent time course experiments. All fits and validations
are available in Supplementary data: Figure 1.
2
1

Figure 3. AKR1D1 catalyses the inactivation of 11KT. AR-activation by 5β-DHT (A) and 11K-5β-
DHT (B). AR activity was measured in HEK293 cells transfected with ARE and a luciferase reporter
construct and treated with conditioned media from incubations of HEK293 cells transiently
transfected with AKR1D1 or empty vector. The activity of the promotor-reporter system was
confirmed by direct treatments with 5α-DHT and 5β-DHT (C). Results represent four independent
experiments performed in triplicate shown as the mean ±SEM. Statistical significance was
determined using a paired t-test with ***, p< 0.001
2
2

Figure 4. The co-expression of AKR1D1 and SRD5A isozymes determine the metabolic fate
of T (A and C) and 11KT (B and D). HEK293 cells expressing AKR1D1 or either SRD5A1 (A and
B) or SRD5A2 (C and D) were mixed at a ratio of 1:1. The resulting 5α- and 5β-reduction of 1 µM
T (A and C) and 11KT (B and D) were measured after 24 hours using UHPSFC-MS/MS.
Experimental results (mean ±SD) are shown in red (5α-product) and blue (5β-product), while the
computational model predictions are shown with black dashed lines.
Figure 5. Computational model of the effect of varying AKR1D1 and SRD5A2 activities.
SRD5A1 activity was fixed while the activity of AKR1D1 and SRD5A2 were varied 10-fold in both
directions. The resulting effects on the 5α- and 5β-reduction of T (blue) and 11KT (red) are shown
as the proportion of 5α-reduced products.
2
3

Figure 6. The sequential production of 11KEt from 11KT. 11KT was 5β-reduced by AKR1D1
expressed in HEK293 cells. Incubation of HEK293 cells expressing AKR1C2 with the resulting 11K-
5β-DHT containing conditioned media subsequently yielded the product 11KEdiol. Further
incubation of HEK293 cells expressing HSD17B2 with the 11KEdiol containing media resulted in
the production of 11KEt. The identity of each product was confirmed by UHPSFC-MS/MS at each
step.
2
4

Figure 7. Formation of Et and 11KEt by HepG2 cells after knockdown of AKR1D1. Relative
mRNA levels of AKR1D1 after knockdown (A). Relative protein levels of AKR1D1 to β-tubulin
after knockdown (B). An (C) and Et (D) and Et:An (E) production from T metabolism in HepG2
cells. 11KAn (F) and 11KEt (G) and 11KEt:11KAn (H) production from 11KT metabolism in
HepG2 cells. Results represent four biological repeats each performed in duplicate shown as the
mean ±SEM. Statistical significance was determined using unpaired t-tests with *, p < 0.05; **, p <
0.005; ***, p < 0.001 and ****, p < 0.0001. SC, scrambled control; KD, knockdown.
2
5

Table 1. Kinetic parameters (Km, app; Vmax, app; Vmax/K ) determined for AKR1D1, SRD5A1 and
m
SRD5A2. N.A., not available. Values in brackets refer to the upper and lower limits of the model
prediction.
Substrate
Product
Vmax, app (µM/h)
Km, app (µM)
Vmax/K
m
T
5β-DHT
0.02 (0.02-0.03)
0.10 (0.05-0.17)
4.09 (2.98-6.34)
0.55 (0.33-0.99)
0.64 (0.23-N.A.)
0.21 (0.17-0.25)
0.24
0.09
0.12
0.01
1.06
AKR1D1
SRD5A1
11KT
11K-5βDHT 0.37 (0.28-0.57)
5α-DHT 0.07 (0.05-0.10)
11K-5α-DHT 0.01 (0.00-N.A.)
5α-DHT 0.22 (0.20-0.25)
T
11KT
SRD5A2 T
2
6

11KT
11K-5α-DHT 0.71 (0.46-1.66)
4.28 (2.59-10.56) 0.17
2
7