5-Bis-(2,6-difluoro-benzylidene) Cyclopentanone Acts as a Selective 11ß-Hydroxysteroid Dehydrogenase one Inhibitor to Treat Diet-Induced Nonalcoholic Fatty Liver Disease in Mice.

Background: 11ß-Hydroxysteroid dehydrogenase one is responsible for activating inert glucocorticoid cortisone into biologically active cortisol in humans and may be a novel target for the treatment of nonalcoholic fatty liver disease. Methods: A series of benzylidene cyclopentanone derivatives were synthesized, and the selective inhibitory effects on rat, mouse and human 11ß-hydroxysteroid dehydrogenase one and two were screened. The most potent compound [5-bis-(2,6-difluoro-benzylidene)-cyclopentanone] (WZS08), was used to treat nonalcoholic fatty liver disease in mice fed a high-fat-diet for 100 days. Results: WZS08 was the most potent inhibitor of rat, mouse, and human 11ß-hydroxysteroid dehydrogenase 1, with half maximum inhibitory concentrations of 378.0, 244.1, and 621.1 nM, respectively, and it did not affect 11ß-hydroxysteroid dehydrogenase two at 100 µM. When mice were fed WZS08 (1, 2, and 4 mg/kg) for 100 days, WZS08 significantly lowered the serum insulin levels and insulin index at 4 mg/kg. WZS08 significantly reduced the levels of serum triglycerides, cholesterol, low-density lipoprotein, and hepatic fat ratio at low concentration of 1 mg/kg. It down-regulated Plin2 expression and up-regulated Fabp4 expression at low concentration of 1 mg/kg. It significantly improved the morphology of the non-alcoholic fatty liver. Conclusion: WZS08 selectively inhibits rat, mouse, and human 11ß-hydroxysteroid dehydrogenase 1, and can treat non-alcoholic fatty liver disease in a mouse model.

Prenatal exposure to di-n-butyl phthalate disrupts the development of adult Leydig cells in male rats during puberty.

Fetal exposure to di-n-butyl phthalate (DBP) causes the adult disease such as lower testosterone production and infertility. However, the mechanism is still unknown. The objective of the present study is to determine how DBP affects the involution of fetal Leydig cells during the neonatal period and how this event causes the delayed development of the adult Leydig cells during puberty. The pregnant Sprague Dawley dams were randomly divided into 3 groups and were gavaged with 0 (corn oil, the vehicle control), 100 or 500mg/kg DBP from gestational day 12 (G12) to G21. The blood and testes were collected from male pups on postnatal day 4 (P4), P7, P14, P21, P28, and P56. Serum testosterone concentrations were assessed and the mRNA levels of Leydig cell- or gonadotroph cell-specific genes were measured. Prenatal exposure to DBP caused the aggregation of fetal Leydig cells, which slowly disappeared when compared to the control. This effect was associated with the reduction of testicular testosterone secretion and down-regulation of the mRNA levels of Leydig cell biomarkers including Scarb1, Star, Cyp11a1, Hsd3b1, Hsd11b1, and Hsd17b3 as well as the gonadotroph biomarkers including Lhb and Gnrhr. In conclusion, we demonstrated that the increased aggregation of fetal Leydig cells by DBP delayed fetal Leydig cell involution, thus leading to the disrupted development of the adult Leydig cells.

Leukemia inhibitory factor stimulates steroidogenesis of rat immature Leydig cells via increasing the expression of steroidogenic acute regulatory protein.

Leukemia inhibitory factor (LIF) has many physiological roles. However, its effects on Leydig cell development are still unclear. Rat immature and adult Leydig cells were cultured with different concentrations of LIF alone or in combination with luteinizing hormone (LH) for 24 h. LIF (1 and 10 ng/ml) significantly increased androgen production in immature Leydig cells, but had no effects on testosterone production in adult Leydig cells. Further studies revealed that LIF dose-dependently increased Star and Hsd17b3 expression levels in immature Leydig cells. Gene microarray revealed that the upregulation of anti-oxidative genes and Star might contribute to LIF-induced androgen production. In conclusion, LIF has stimulatory effects on androgen production in rat immature Leydig cells.

Gossypol enantiomers potently inhibit human placental 3ß-hydroxysteroid dehydrogenase 1 and aromatase activities.

Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 µM, while (-) gossypol weakly inhibited it with IC50 over 100 µM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 µM, while (+) gossypol had no inhibition when the highest concentration (100 µM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects.

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells.

BACKGROUND: Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3ß-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. METHODOLOGY: Immature Leydig cells isolated from 35 day-old rats were cultured with 30 µM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 µM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. RESULTS AND CONCLUSIONS: In intact Leydig cells, 30 µM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 µM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 µM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 µM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.

Determination of songorine in rat plasma by UPLC-MS/MS: Assay development and application to pharmacokinetic study.

An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of songorine in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2mL of acetonitrile to a 0.1mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0min and the elution of songorine was at 1.68min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 358.3→340.3 for songorine and m/z 237.2→194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1-1000ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of songorine in plasma was in the range of 75.2-87.5%. The intra- and inter-day precision (RSD) was between 3.1-8.5% and 4.3-9.6% and the intra- and inter-day accuracy (RE) ranged from -4.0 to 8.9% and -9.0 to 6.7%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0mg/kg songorine in rats.

Zearalenone Inhibits Rat and Human 11ß-Hydroxysteroid Dehydrogenase Type 2.

Zearalenone is a mycotoxin produced by Fusarium spp. 11ß-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local level of active glucocorticoid, which has a broad range of physiological actions. In the present study, the potency of zearalenone was tested for the inhibition of HSD11B1 and HSD11B2 in rat and human tissues. Zearalenone showed potent inhibition of HSD11B2 with the half-maximal inhibitory concentration (IC50) calculated at 49.63 and 32.22 µM for the rat and human, respectively. Results showed that zearalenone competitively inhibited HSD11B2 when a steroid substrate was used. However, it served as an uncompetitive inhibitory factor when the cofactor NAD(+) was used. In contrast, the potency of zearalenone to inhibit both rat and human HSD11B1 was diminished, with the concentration of 100 µM causing almost no inhibitory effect on the isoform. In conclusion, we observed that zearalenone is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as kidney and placentas.