11β-Hydroxysteroid Dehydrogenase 2 in Rat Leydig Cells: Its Role in Blunting Glucocorticoid Action at Physiological Levels of Substrate

Abstract

Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.

Distribution of glucocorticoid receptors and 11 beta-hydroxysteroid dehydrogenase isoforms in the rat inner ear

“…After being washed 3 times in PBS, the sections were autoclaved in 10mM citrate buffer for 10 min at 121℃. antibodies showed a single band of the expected size in western blot analysis and the specificity was confirmed in previous other studies (Brewer JA, et al, 2002;Ge RS, et al, 2005;Honda, et al, 2008). The sections were incubated with the first antibody of anti-GR (1:200), anti-11β-HSD1 (1:100), and anti-11β-HSD2 (1:100) with 5% BSA in PBS for overnight, washed 4 times with 0.075% Brij35 (B4184, Sigma-Aldrich, St.…”

Rapid mechanisms of glucocorticoid signaling in the Leydig cell☆

“…When 11βHSD oxidative and reductive activities are measured in freshly isolated Leydig cells, the oxidative activity exceeds the reductive activity by a 2:1 ratio that is the inverse of the ratio measured in freshly isolated hepatocytes assayed in parallel under identical conditions [45]. This primary oxidative activity in rat Leydig cells may be not contributed by 11βHSD2 since it has only one thousandth of 11βHSD1 in this cell type [50].…”

Effect of intramuscular injection of butafosfan and cobalamin on the quality of Fresh and Cooled Stallion Semen

“…Both cobalamin and phosphorus participate in the function of enzymes that modulate the intracellular concentrations of glucocorticoids, maintaining a balance between the active and inactive forms, cortisone and cortisol respectively (GE et al, 2005;SHARP;WITTS, 1962). Therefore, butafosfan and cobalamin could have a protective effect on testicular function in stallions in stressful situations or on prolonged treatment with steroids.…”