Identification of a Kinetically Distinct Activity of 11β-Hydroxysteroid Dehydrogenase in Rat Leydig Cells¹

Abstract

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11beta-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11beta-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11beta-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11beta-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11beta-HSD and MR messenger RNAs (mRNAs), and for type I 11beta-HSD protein. Leydig cell 11beta-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11beta-HSD is present in rat Leydig cells. However, unlike the type I 11beta-HSD in liver parenchymal cells, the Leydig cell 11beta-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 +/- 9.3 nM and maximum velocity (Vmax) of 7.1 +/- 1.2 pmol x min x 10(6) cells. The second component consisted of high Km activities that were consistent with type I:NADP-dependent oxidative activity with Km of 5.87 +/- 0.46 microM and Vmax of 419 +/- 17 pmol x min x 10(6) cells, and NADPH-dependent reductive activity with Km of 0.892 +/- 0.051 microM and Vmax of 117 +/- 6 pmol x min x 10(6) cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11beta-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11beta-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.

Benzofuran Derivatives Inhibit 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Rat Adipose Tissue

“…The apparent K m value for 11-dehydrocorticosterone of 11β-HSD1 in rat adipose tissue microsomes (0.64 µM) were approximated to the values reported by others, which were 0.25 µM 26) and 0.89 µM 27) in rats. In more recent years, considerable activity in the pharmaceutical industry has led to the discovery of several classes of 11β-HSD1 inhibitors, 12,13) such as thiazoles, adamantane amides, diarylsulfonamides, etc., and many compounds and a variety of natural products have been reported as candidates for selective 11β-HSD1 inhibitors.…”

Relationship between ovarian cortisol:cortisone ratios and the clinical outcome of in vitro fertilization and embryo transfer (IVF‐ET)

“…However, after exposure to surge concentrations of gonadotrophins, 11βHSD2 expression is suppressed and the luteinizing human granulosa cells switch to expression of the type 1 11βHSD, assumed to function predominantly as an 11‐ketosteroid reductase (Tetsuka et al ., 1997). In addition, functionally luteinized human granulosa cells also appear to express a biochemically distinct, high affinity, NADP + ‐dependent 11βHSD activity (Michael et al ., 1997) comparable to that described in rat testis Leydig cells (Gao et al ., 1997; Ge et al ., 1997). Hence, multiple isoforms of 11βHSD will interact to dictate the ratio of cortisol:cortisone in FF aspirated from follicles at different stages of development.…”

Insights into the Development of the Adult Leydig Cell Lineage from Stem Leydig Cells

“…The directions are regulated by another enzyme, hexose-6-phosphate dehydrogenase. Since this regulatory enzyme is very low in ALCs (Ge et al, 1997a; Li et al, 2015) HSD11B1 mainly functions as an oxidase to metabolize glucocorticoid into an inactive form. Also, in rat ALCs, a CYP2A1 begins to express and it can catalyze testosterone into 7α-hydroxytestosterone, which also involves in the regulation of HSD11B1 activities (Hu et al, 2010a).…”