Syed A. Latif

The effects of the licorice derivative, glycyrrhetinic acid, on hepatic 3α- and 3β-hydroxysteroid dehydrogenases and 5α- and 5β-reductase pathways of metabolism of aldosterone in male rats

Abstract Ingestion of licorice or treatment with chemical derivatives of glycyrrhetinic acid (GA), an active principle of licorice, can cause hypertension, sodium retention, and hypokalemia. Although GA has been shown to inhibit 11β-hydroxy steroid dehydrogenase, it may not be the only hepatic enzyme affected by this licorice derivative. Therefore, we studied the effects of GA on other major hepatic steroid-metabolizing enzymes from adrenalectomized male rats using aldosterone as the substrate; namely, Δ4-5α and Δ4-5β-reductases and 3α- and 3β-hydroxysteroid dehydrogenases (3α- and 3β-HSD). From these in vitro studies, we demonstrated that GA does not affect either microsomal 5α-reductase or cytosolic 3α-HSD activity. However, GA is a potent inhibitor of cytosolic 5β-reductase: the K(inis) and k(inii) were calculated from enzyme kinetic analysis to be 6.79 and 5.41 μm, respectively, using the Cleland equation, indicating that GA is a noncompetitive inhibitor of aldosterone. In addition, GA specifically inhibited microsomal 3β-HSD enzyme activity by what appears to be a competitive inhibition mechanism, causing a build-up of the intermediate, 5α-dihydroaldosterone (DHAldo). Thus, this study has indicated that GA has a profound effect on hepatic ring A-reduction of aldosterone. Inhibition of 5β-reductase and 3β-HSD results in decreased synthesis of both 3α, 5β-tetrahy-droaldosterone (THAldo) and 3β, 5α-THAldo and, hence, accumulation of aldosterone and 5α-DHAldo, both potent mineralocorticoids. Thus, more than one enzyme may be involved in the mechanism by which GA causes steroids, such as the glucocorticoid hormones, to display mineralocorticoid-like actions in humans and experimental animals. (Steroids 55:52–58, 1990)

Rapid mechanisms of glucocorticoid signaling in the Leydig cell

Stress-mediated elevations in circulating glucocorticoid levels lead to corresponding rapid declines in testosterone production by Leydig cells in the testis. In previous studies we have established that glucocorticoids act on Leydig cells directly, through the classic glucocorticoid receptor (GR), and that access to the GR is controlled prior to the GR by a metabolizing pathway mediated by the type 1 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD1). This enzyme is bidirectional (with both oxidase and reductase activities) and in the rat testis is exclusively localized in Leydig cells where it is abundantly expressed and may catalyze the oxidative inactivation of glucocorticoids. The predominant reductase direction of 11betaHSD1 activity in liver cells is determined by an enzyme, hexose-6-phosphate dehydrogenase (H6PDH), on the luminal side of the smooth endoplasmic reticulum (SER). Generation of the pyridine nucleotide cofactor NADPH by H6PDH stimulates the reductase direction of 11betaHSD1 resulting in increased levels of active glucocorticoids in liver cells. Unlike liver cells, steroidogenic enzymes including 17beta-hydroxysteroid dehydrogenase 3 (17betaHSD3) forms the coupling with 11betaHSD1. Thus the physiological concentrations of androstenedione serve as a substrate for 17betaHSD3 utilizing NADPH to generate NADP+, which drives 11betaHSD1 in Leydig cells primarily as an oxidase; thus eliminating the adverse effects of glucocorticoids on testosterone production. At the same time 11betaHSD1 generates NADPH which promotes testosterone biosynthesis by stimulating 17betaHSD3 in a cooperative cycle. This enzymatic coupling constitutes a rapid mechanism for modulating glucocorticoid control of testosterone biosynthesis. Under stress conditions, glucocorticoids also have rapid actions to suppress cAMP formation thus to lower testosterone production.

Stimulation of testosterone production in rat Leydig cells by aldosterone is mineralocorticoid receptor mediated.

The testis is known to be a site of corticosterone action, and testosterone production in Leydig cells is directly inhibited by glucocorticoids. Glucocorticoids bind to both glucocorticoid receptors (GRs) and to mineralocorticoid receptors (MRs). In Leydig cells, selective mineralocorticoid binding could result from oxidative inactivation of glucocorticoid by type 1 and/or 2 11beta-hydroxysteroid dehydrogenase (11betaHSD), as both isoforms are expressed. However, it remains unclear whether Leydig cells express MRs and respond directly to mineralocorticoid action. Therefore, the aims of the present study were to ascertain: (1) whether MR mRNA, protein and receptor binding are present in Leydig cells; and (2) if the mineralocorticoid modulates testosterone production. The mRNA encoding MR, as well as protein, and binding activity were each observed in adult rat Leydig cells. MR-ligand binding specificity within isolated Leydig cells was evaluated further by measuring displacement of MR binding to aldosterone by corticosterone in the presence and absence of carbenoxolone, an inhibitor of 11betaHSD1 and 2 that decreases conversion to biologically inert 11-dehydrocorticosterone. Carbenoxolone inhibited 11betaHSD oxidative activity, and reduced corticosterone-binding by 50%. Mineralocorticoid effects on steroidogenesis were assessed in the presence of aldosterone (0.01-10 nM) with or without the MR antagonist, RU28318. Aldosterone induced dose-dependent increases in both basal and luteinizing hormone-stimulated testosterone production. RU28318 eliminated the increase, indicating that these effects of aldosterone were mediated by the MR. The effects of aldosterone and luteinizing hormone (0.1 ng/ml) on testosterone production were synergistic, suggesting that the two hormones increased steroidogenesis through separate pathways. We conclude that Leydig cells express MRs and that testosterone production is subject to regulation by aldosterone.

Endogenous selective inhibitors of 11β-hydroxysteroid dehydrogenase isoforms 1 and 2 of adrenal origin

In earlier studies [Latif, S.A., Sheff, M.F., Ribeiro, C.E., Morris, D.J., 1997. Selective inhibition of sheep kidney 11beta-hydroxysteroid-dehydrogenase isoform 2 activity by 5alpha-reduced (but not 5beta) derivatives of adrenocorticosteroids. Steroids 62, 230-237], only derivatives of steroid hormones possessing the 5alpha-Ring A-reduced configuration selectively inhibited 11beta-HSD2-dehydrogenase, whereas their 5beta-derivatives were inactive. This present study focuses on an expanded group of endogenous 11-oxygenated, 5alpha and 5beta-Ring A-reduced metabolites of adrenocorticosteroids, and progestogen and androgen steroid hormones. These substances were tested for their inhibitory properties against 11beta-HSD2, 11beta-HSD1-dehydrogenase and 11beta-HSD1 reductase. The present studies showed that the following compounds stand out as potent inhibitors. These are 5alpha-DH-corticosterone, 3alpha,5alpha-TH-corticosterone, 11beta-OH-progesterone, 11beta-OH-allopregnanolone, 11beta-OH-testosterone, and 11beta-OH-androstanediol, inhibitors of 11beta-HSD1-dehydrogenase; 3alpha,5alpha-TH-11-dehydro-corticosterone, 11-keto-progesterone, 11-keto-allopregnanolone, and 11-keto-3beta,5alpha-TH-testosterone, inhibitors of 11beta-HSD1 reductase; 3alpha,5alpha-TH-aldosterone, 5alpha-DH-corticosterone, 3alpha,5alpha-TH-corticosterone,11-dehydro-corticosterone, 3alpha,5alpha-TH-11-dehydro-corticosterone, 11beta-OH-progesterone, 11-keto-progesterone, 11beta-OH-allopregnanolone, 11-keto-allopregnanolone, 11beta-OH-testosterone, and 11-keto-testosterone, inhibitors of 11beta-HSD2. All of these substances have the potential to be derived from adrenally synthesized corticosteroids. Substances with similar structures to those described may help in the design of exogenous agents for the management of a variety of disease states involving 11beta-HSD isoenzymes.

Expression of 11β-Hydroxylase in Rat Leydig Cells

11β-Hydroxy (11β-OH) derivatives of certain steroids function as inhibitors of 11β-hydroxysteroid dehydrogenase isoform 1 (11βHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11β-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11β-hydroxylation of steroids and thus producing 11β-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11β-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11β-hydroxylated steroids inhibit Leydig cell 11βHSD1 activities. 11β-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expressi...

Possible endogeneous regulators of steroid inactivating enzymes and glucocorticoid-induced Na+ retention

Various endogenous substances which bear similar structural resemblances to glycyrrhetininc acid were screened for inhibitory activity against 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) and 5 beta-reductase (5 beta-R). Among the compounds screened, 3 alpha, 5 beta-tetrahydroprogesterone (3 alpha,5 beta-THP) was a potent inhibitor of 11 beta-OHSD and a moderate inhibitor of 5 beta-R. Of the bile acids tested, chenodeoxycholic acid (CDCA) was the most potent inhibitor of both 11 beta-OHSD and 5 beta-R. Cholic acid (CA), a moderate inhibitor of 11 beta-OHSD was a weak inhibitor of 5 beta-R, whereas deoxycholic acid was a moderate inhibitor of 5 beta-R but a weak inhibitor of 11 beta-OHSD. 3 alpha, 5 beta-THP and bile acids were also tested to determine whether, like GA, they could confer mineralocorticoid actions upon corticosterone (B). In adrenalectomized rats pretreated with CDCA or 3 alpha,5 beta-THP, B caused a significant antinatriuresis; the effect of B plus CDCA was blocked by the antimineralocorticoid, RU 28318. Thus, we report on two structurally similar endogenous substances, 3 alpha, 5 beta-THP and CDCA, which inhibit both 11 beta-OHSD and 5 beta-R activity, and which can confer mineralocorticoid actions upon the glucocorticoid, B.

Selective inhibition of sheep kidney 11β-hydroxysteroid dehydrogenase isoform 2 activity by 5α-reduced (but not 5β) derivatives of adrenocorticosteroids

Abstract We have previously reported that 5α and 5β pathways of steroid metabolism are controlled in vivo by dietary Na + and glycyrrhetinic acid, see Gorsline et al. 1998; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11β-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11β-HSD2 (kidney). The activity of 11β-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11β-HSD2 is particularly affected. In all three conditions, excretion of the less common 5α metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5α and 5β) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP + as co-factor (11β-HSD1), and sheep kidney microsomes with NAD + as co-factor (11β-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11β-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5α and 5β derivatives. 11β-HSD1 is inhibited by both 5α and certain 5β derivatives. 11β-HSD-2 was selectively inhibited only by 5α derivatives; 5β derivatives were without inhibitory activity toward this isoform of 11β-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11β-HSD1 and 11β-HSD2.

The metabolism of aldosterone in kidney

Slices of kidney cortex and medulla from adrenalectomized male rats metabolized aldosterone to at least 4 peaks of polar metabolites as well as reduced metabolites. The majority of the products were ring A-reduced and shown chromatographically to consist of 5 alpha-reduced metabolites; significant quantities of 5 beta-reduced metabolites were also synthesized. Approximately half of the 5 alpha-reduced products cochromatographed with 3 beta,5 alpha-THA and the remainder with 5 alpha-DHA and/or 3 alpha,5 alpha-THA. The anti-mineralocorticoids, spironolactone and progesterone, inhibited renal synthesis of 5 alpha-reduced products of aldosterone by 80%. 5 beta-Reduction was also slightly inhibited. Corticosterone slightly inhibited synthesis of the 5 alpha- and 5 beta-reduced products. Corticosterone and progesterone significantly inhibited the renal synthesis of the polar metabolites of aldosterone, but the inhibition was not significant at this dosage of spironolactone. Interestingly, the nuclei and plasma membranes were shown to be the most active fractions for these aldosterone transformations. Importantly significant quantities of 5 alpha-DHA and/or 3 alpha,5 alpha-THA, which possess 1/10 and 1/30, respectively, of the antinatriuretic activity of aldosterone, were synthesized at these subcellular locations.

Effects of carbenoxolone administered acutely to adrenalectomized rats (in vivo) on renal and hepatic handling of corticosterone by 11β-hydroxysteroid dehydrogenase

The in vivo effect(s) of carbenoxolone (CS) on renal 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD), hepatic 11 beta-OHSD, and 5 beta-reductase enzymatic activity was investigated, under conditions previously shown to confer mineralocorticoid (MC)-like activity on the glucocorticoids cortisol and corticosterone; it has been suggested that this Na+ retention is linked to inhibition of renal 11 beta-OHSD. The results show that acute administration of CS [2.5 mg/rat for 0.5 or 2 hours; and 10 or 25 mg/rat for 2 hours subcutaneously (sc)] to rats caused no inhibition of 11 beta-OHSD activity in kidney homogenates, minces, and microsomes when compared with controls. However, addition of 50 nM CS to the incubation medium completely inhibited the 11 beta-OHSD activity in kidney homogenates and microsomes (from controls or CS-injected rats). In contrast, hepatic microsomal 11 beta-OHSD was significantly inhibited after in vivo treatment with CS (P < 0.05) using 2 microM and 50 microM corticosterone, as was 5 beta-reductase (P < 0.05) using 4 microM corticosterone as substrate. However, chronic glycyrrhizin administration (15 mg/rat/day sc for 14 days) significantly inhibited renal 11 beta-OHSD activity when assayed in minces or homogenates. Thus, it appears that when CS is administered acutely, its effects are primarily on hepatic 11 beta-OHSD and 5 beta-reductase with no inhibition of renal 11 beta-OHSD.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: Effects of antimineralocorticoids

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.