Cell experiment:

AML-12 cells are used and cultured in a 1:1 mix of DMEM/Ham’s F12 medium supplemented with 10% fetal bovine serum, 1% insulin-transferrin-selenium mix, 40 ng/mL dexamethasone, 100 U/mL penicillin, and 100 μg/mL streptomycin. For gene regulation studies, AML-12 cells are plated in growth medium at 4×105 cells per well in six-well plates. The following day, treatments including Androsterone or dimethylsulfoxide (DMSO) vehicle are added to the medium. At various times after treatment addition, total RNA is prepared. mRNA levels for individual genes are determined by real-time PCR[1].

Animal experiment:

Castrated male mice 8 to10 wk of age are fed a casein diet for 2 wk before the start of the study. The mice receive daily sc injections of 0.1 mL 90% corn oil/10% ethanol vehicle or 10 mg/mL Androsterone in vehicle. Animals are killed 2 h after the fifth daily treatment, approximately 4 h after commencement of the light cycle. Total RNA is prepared, and mRNA levels for specific genes are determined by real-time PCR[1].

Androsterone is a metabolic product of testosterone and can activate Farnesoid X Receptor (FXR).

Androsterone activates both the mFXR-LBD and the hFXR-LBD, with Androsterone activating the mFXR-LBD more strongly than the hFXR-LBD. Furthermore, cotransfection studies with gal4-hFXR-LBD and SRC-1/VP16 expression plasmids demonstrate that Androsterone potentiates the interaction of SRC-1 with the hFXR-LBD. Several amino acid changes including H294S, S332V, R351H, and Y361F significantly reduce Androsterone activation[1]. Androsterone (5α, 3α-A) (10 to 100 μM) also inhibits epileptiform discharges in a concentration-dependent fashion in the in vitro slice model[2].

Androsterone treatment results in a significant induction of small heterodimer partner (SHP), suggesting Androsterone may activate endogenous FXR[1]. Intraperitoneal injection of Androsterone (5α, 3α-A) protects mice in a dose-dependent fashion from seizures in the following models (ED50, dose in mg/kg protecting 50% of animals): 6 Hz electrical stimulation (29.1), pentylenetetrazol (43.5), pilocarpine (105), 4-AP (215), and maximal electroshock (224)[2].

[1]. Wang S, et al. The nuclear hormone receptor farnesoid X receptor (FXR) is activated by androsterone. Endocrinology. 2006 Sep;147(9):4025-33. [2]. Kaminski RM, et al. Anticonvulsant activity of androsterone and etiocholanolone. Epilepsia. 2005 Jun;46(6):819-27.