Metabolic Coupling Determines the Activity: Comparison of 11ß-Hydroxysteroid Dehydrogenase 1 and Its Coupling between Liver Parenchymal Cells and Testicular Leydig Cells.

BACKGROUND: 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) interconverts active 11ß-hydroxyl glucocorticoids and inactive 11keto forms. However, its directionality is determined by availability of NADP+/NADPH. In liver cells, 11ß-HSD1 behaves as a primary reductase, while in Leydig cells it acts as a primary oxidase. However, the exact mechanism is not clear. The direction of 11ß-HSD1 has been proposed to be regulated by hexose-6-phosphate dehydrogenase (H6PDH), which catalyzes glucose-6-phosphate (G6P) to generate NADPH that drives 11ß-HSD1 towards reduction. METHODOLOGY: To examine the coupling between 11ß-HSD1 and H6PDH, we added G6P to rat and human liver and testis or Leydig cell microsomes, and 11ß-HSD1 activity was measured by radiometry. RESULTS AND CONCLUSIONS: G6P stimulated 11ß-HSD1 reductase activity in rat (3 fold) or human liver (1.5 fold), but not at all in testis. S3483, a G6P transporter inhibitor, reversed the G6P-mediated increases of 11ß-HSD1 reductase activity. We compared the extent to which 11ß-HSD1 in rat Leydig and liver cells might be coupled to H6PDH. In order to clarify the location of H6PDH within the testis, we used the Leydig cell toxicant ethane dimethanesulfonate (EDS) to selectively deplete Leydig cells. The depletion of Leydig cells eliminated Hsd11b1 (encoding 11ß-HSD1) expression but did not affect the expression of H6pd (encoding H6PDH) and Slc37a4 (encoding G6P transporter). H6pd mRNA level and H6PDH activity were barely detectable in purified rat Leydig cells. In conclusion, the availability of H6PDH determines the different direction of 11ß-HSD1 in liver and Leydig cells.

An alternative explanation of hypertension associated with 17α-hydroxylase deficiency syndrome.

The syndrome of 17α-hydroxylase deficiency is due to the inability to synthesize cortisol and is associated with enhanced secretion of both corticosterone and 11-deoxy-corticosterone (DOC). In humans, corticosterone and its 5α-Ring A-reduced metabolites are excreted via the bile into the intestine and transformed by anaerobic bacteria to 21-dehydroxylated products: 11ß-OH-progesterone or 11ß-OH-(allo)-5α-preganolones (potent inhibitors of 11ß-HSD2 and 11ß-HSD1 dehydrogenase). Neomycin blocks the formation of these steroid metabolites and can blunt the hypertension in rats induced by either ACTH or corticosterone. 3α,5α-Tetrahydro-corticosterone, 11ß-hydroxy-progesterone, and 3α,5α-tetrahydro-11ß-hydroxy-progesterone strongly inhibit 11ß-HSD2 and 11ß-HSD1 dehydrogenase activity; all these compounds are hypertensinogenic when infused in adrenally intact rats. Urine obtained from a patient with 17α-hydroxylase deficiency demonstrated markedly elevated levels of endogenous glycyrrhetinic acid-like factors (GALFs) that inhibit 11ß-HSD2 and 11ß-HSD1 dehydrogenase activity (>300 times greater, and >400 times greater, respectively, than those in normotensive controls). Thus, in addition to DOC, corticosterone and its 5α-pathway products as well as the 11-oxygenated progesterone derivatives may play a previously unrecognized role in the increased Na(+) retention and BP associated with patients with 17α-hydroxylase deficiency.

Role of 11ß-OH-C(19) and C(21) steroids in the coupling of 11ß-HSD1 and 17ß-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells.

Here we describe further experiments to support our hypothesis that bidirectional 11ß-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17ß-HSD3, incubation of Leydig cells with corticosterone (B) or several C(19)- and C(21)-11ß-OH-steroids, in the presence of [(3)H]-11-dehydro-corticosterone (A), stimulated 11ß-HSD1-reductase activity. However, in presence of 30 µM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11ß-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p<0.05) to 224-267 picomole/25,000 cells/30 min when 10 µM 11ß-OH-steroids (in addition to 30 µM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 µM), and B or A (500 nM). Incubations of 0.3-6.0 µM of corticosterone (plus or minus 30 µM AD) were then performed to test the effectiveness of 17ß-HSD3 as a possible NADP(+) regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 µM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11ß-HSD1 is enzymatically coupled to 17ß-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.

Interactions of mineralocorticoids and glucocorticoids in epithelial target tissues revisited.

The interplay between mineralocorticoids (MCs) and glucocorticoids (GCs) in sodium transporting epithelia is complex and only partially understood. In seminal papers published in the years soon after the discovery of aldosterone, various investigators experimentally observed that mineralocorticoid-induced renal sodium retention could only be reliably measured in adrenalectomized animals. Addition of endogenous GCs or their 11-dehydro metabolites blunted the antinatriuretic action of aldosterone and 11-dehydro-GCs decreased binding of aldosterone to mineralocorticoid receptors (MR). Under normal circumstances, endogenous GCs alone do not induce sodium transport in MC responsive epithelia yet these same GCs are able to activate MR and induce sodium transport if the enzyme 11beta-HSD2 is inhibited. Given the physiologic concentrations of both MCs and GCs, it is likely that the local epithelial cell exposure to GCs is great enough to allow GC binding to MR despite the presence of 11beta-HSD2. Thus other factors supplement the receptor selectivity role suggested for 11beta-HSD2. Why GCs bind to MR under one set of conditions and produce no effect and under different sets of conditions (11beta-HSD2 inhibition) elicit sodium transport remains a puzzle to be solved. What is clear is that a dual role for 11beta-HSD2 is emerging; first as the putative "guardian" over the MR reducing GC binding, and second as a source for 11-dehydro-GCs, which may serve as endogenously and locally produced "spironolactone-like substances", which may thus attenuate aldosterone-induced sodium transport.

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.

Correlation of glycyrrhetinic acid-like factors (kidney 11beta-HSD2-GALFs) with urinary free cortisol and plasma renin activity in essential hypertension.

Previously we reported that urinary levels of glycyrrhetinic acid-like factors (11beta-HSD2-GALFs) were increased in a subset of patients with essential hypertension when maintained on a low-Na(+) diet. The present studies were undertaken to correlate changes in urinary GALF levels with urinary free cortisol (UFC) and plasma renin activity (PRA). The amounts of GALFs markedly increased from 7.38 +/- 0.80 to 14.58 +/- 1.94 (P < .0003) in the high/normal renin and from 5.60 +/- 0.77 to 8.39 +/- 1.08 (P < .045) in the low renin patients on a low-Na(+) diet compared with high-Na(+) diet with no effect in the normotensive controls (P < .668). The elevated GALF levels in high/normal renin hypertensives maintained on the low-Na(+) diet strongly correlated with the increased UFC levels and also with PRA; no such correlations were observed with either the normotensive controls or low renin hypertensives. In high/normal renin hypertensives, the elevated 11beta-HSD2-GALFs may have two major functions: increased Na(+) retention by the kidney by allowing cortisol to access the renal mineralocorticoid receptor and increased vascular reactivity by allowing cortisol to access the vascular mineralocorticoid receptor.

Endogenous selective inhibitors of 11beta-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.

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.

11{beta}-Hydroxysteroid dehydrogenase 2 in rat leydig cells: its role in blunting glucocorticoid action at physiological levels of substrate.

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.

Effect of chenodeoxycholic acid on 11beta-hydroxysteroid dehydrogenase in various target tissues.

Glucocorticoids are metabolized by isoforms of the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). There is some controversy concerning the bile acid, chenodeoxycholic acid (CDCA), as a potential endogenously produced inhibitor of 11beta-HSD. The present experiments were designed to determine the relative specificity of CDCA for both isoforms of 11beta-HSD and to assess the biological relevance of inhibition in vascular tissue. IC(50) values (concentrations which inhibit 50% of the enzyme reaction) were calculated using rat liver microsomes as a source of 11beta-HSD1 dehydrogenase, Leydig cells for 11beta-HSD1 dehydrogenase and reductase, aorta for 11beta-HSD1 dehydrogenase and reductase, and sheep kidney for 11beta-HSD2 dehydrogenase. In each case, CDCA functioned as a potent inhibitor of 11beta-HSD1 dehydrogenase with IC(50) values of ranging from 0.2 to 7 micromol/L in contrast to 37 to 200 micromol/L for 11beta-HSD1 reductase. CDCA exhibited relatively weak inhibitory activity against 11beta-HSD2 from sheep kidney with an IC(50) of 70 micromol/L. The effect of CDCA on vascular contraction was studied in aortic rings isolated from Spague-Dawley rats incubated in medium containing corticosterone 10 nmol/L +/- CDCA (1 micromol/L) for 24 hours. Rings were stimulated with graded concentrations of phenylephrine (PE) (10 nmol/L, 100 nmol/L, and 1 micromol/L). Rings exposed to corticosterone and CDCA consistently demonstrated a greater contractile response at lower doses of PE (63% at PE 10 nmol/L, P <.001; 20% at PE 100 nmol/L, P <.025; and 10% at PE 1 micromol/L, not significant [NS]) compared to control preparations incubated with cortiosterone alone. These studies demonstrate (1) that CDCA preferentially affects 11beta-HSD1 dehydrogenase; (2) CDCA does inhibit 11beta-HSD2 dehydrogenase and 11beta-HSD1 reductase but only at high(er) concentrations exceeding 70 micromol/L and 37 micromol/L, respectively; and (3) inhibition of 11beta-HSD1 dehydrogenase in aortic rings by CDCA (1 micromol/L) enhances the contractile response of corticosterone plus PE.

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-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 expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.