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Published-Ahead-of-Print January 9, 2008, DOI:10.2164/jandrol.107.004242
Journal of Andrology, Vol. 29, No. 3, May/June 2008
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.107.004242

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Inhibition of 11β-Hydroxysteroid Dehydrogenase Enzymatic Activities by Glycyrrhetinic Acid In Vivo Supports Direct Glucocorticoid-Mediated Suppression of Steroidogenesis in Leydig Cells

GUO-XIN HU*, HAN LIN{dagger},{ddagger}, CHANTAL M. SOTTAS{ddagger}, DAVID J. MORRIS§, MATTHEW P. HARDY|| AND REN-SHAN GE*,{ddagger}

From the * Institute of Molecular Toxicology and Pharmacology, School of Pharmacy, and the {dagger} Institute of Neuroendocrinology and 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, People's Republic of China; the {ddagger} Population Council and The Rockefeller University, New York, New York; and the § Department of Pathology and Laboratory Medicine, The Miriam Hospital, Brown University School of Medicine, Providence, Rhode Island.

Correspondence to: Dr Ren-Shan Ge, The Population Council, 1230 York Avenue, New York, NY 10021 (e-mail: rge{at}popcbr.rockefeller.edu).
Received for publication September 25, 2007; accepted for publication January 9, 2008.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Previous studies have suggested that glucocorticoid (GC) can directly affect testicular testosterone (T) biosynthesis by Leydig cells through a receptor-mediated mechanism. Interconversion of corticosterone (CORT), the active form in rodents, and 11-dehydroCORT, the biologically inert 11-keto form, is catalyzed by 11βHSD1. This enzyme thus controls the intracellular concentration of active GC. We have postulated that elevated CORT levels resulting from stress exceed the Leydig cell's capacity for metabolic inactivation of CORT, resulting in suppressed T production. The present study tested whether inhibition of 11βHSD1 in vivo, by the administration of glycyrrhetinic acid (GA), increases intracellular active GC concentration and thereby affects serum T concentration and Leydig cell T production. Adult Sprague-Dawley rats were treated with vehicle (corn oil), CORT, GA, or GA + CORT. Serum luteinizing hormone (LH), CORT, and T levels were measured, as were the steroidogenic capacities of purified Leydig cells. Twofold elevations of CORT were achieved by the administration of either CORT or GA alone, but in both cases there was no effect on serum T levels. However, when CORT and GA were administered in combination, serum CORT levels increased 3.5-fold (to 420 ± 34 ng/mL) and serum T levels were reduced significantly (to 0.72 ± 0.07 ng/mL; control, 2.12 ± 0.23 ng/mL). Serum levels of LH were not affected by CORT, GA, or GA + CORT. Consistent with the reduced serum T levels following GA + CORT, steroidogenic enzyme expression and capacities were significantly reduced compared to control. These data support a role for 11βHSD1 in modulating intracellular CORT concentrations and, in turn, for a direct effect of GC on Leydig cells in response to stress.

     Key words: CORT, testosterone


In mammalian species, glucocorticoids (GCs) induce a variety of responses in cells, including proliferation, differentiation, and apoptosis, via the GC receptor (GR) (Schmid et al, 1995). Effects of GCs are exerted through their receptor proteins, GRs, which, when bound to ligand, associate with specific DNA sequences, termed GC response elements (GREs). The GREs are present in the promoter regions of target genes, and mediate either increased or repressed transcription (Landers and Spelsberg, 1992). In addition, GRs can interfere with the transcriptional activity of factors such as the AP-1 complex by direct protein-to-protein interactions (Jonat et al, 1990). In rats, increases in corticosterone (CORT) concentrations have been shown to accompany exposure to stressors such as restraint (Orr and Mann, 1992) and psychosocial interaction with a dominant male (Monder et al, 1994). Leydig cells are known to express GRs and to respond to GC (Stalker et al, 1989; Schultz et al, 1993; Ge et al, 1997). It has been suggested by some studies that increased CORT induced by stress might result in direct inhibition of testosterone (T) production by Leydig cells through a GR-dependent mechanism (Orr and Mann, 1992; Monder et al, 1994a; Gao et al, 1996), independent of stress-induced changes in circulating levels of luteinizing hormone (LH) (Gao et al, 1996) or neurotransmitters (Kostic et al, 1998b; Weissman et al, 2007).

The 11βHSD1 enzyme catalyzes the interconversion of active GCs and their biologically inert 11-keto metabolites, and has been shown to be an important determinant of GC bioactivity (Monder and White, 1993). Rat Leydig cells contain bidirectional 11βHSD activity, with oxidase activity predominant (Gao et al, 1997; Ge et al, 1997). To address the hypothesis that 11βHSD activity plays a role in ameliorating the suppressive effects of GCs on Leydig cell steroidogenesis, we administered glycyrrhetinic acid (GA), a bidirectional inhibitor of the enzyme, and/or CORT to rats. The rationale for administering CORT was to potentially override the capacity of 11βHSD oxidase activity in Leydig cells in vivo, thus resulting in increased intracellular availability of active GC. We show that when rats were administered GA + CORT, circulating CORT concentrations increased over 3.5-fold, and Leydig cell steroidogenesis was inhibited significantly. The decline in androgen production was associated with diminished expression levels of genes that encode proteins required for steroidogenesis.


   Materials and Methods
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Results
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Chemicals

[1,2,-3H]CORT (specific activity, 40 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, Massachusetts). [1,2,-3H]11dehydroCORT (11DHC) was prepared from labeled CORT as described earlier (Lakshmi and Monder, 1985). Cold CORT, 11DHC, and T were purchased from Steraloids (Wilton, New Hampshire). GA was purchased from Sigma Co (Ronkonkoma, New York).

Animals and Treatment

Male Sprague-Dawley rats (250–300 g) were purchased from Charles River Laboratories (Wilmington, Massachusetts). Rats were divided into 4 groups (18 rats per group), with rats of each group receiving 3 intraperitoneal injections at 0, 12, and 24 hours as follows: 1) vehicle control (corn oil and DMSO:water [1:4, vol/vol]); 2) CORT alone (10 mg/kg each injection); 3) GA alone (5 mg/kg each injection); and 4) GA + CORT. One hour after the last injection, rats were killed by asphyxiation with CO2. Blood samples were centrifuged at 500 x g, and the sera were stored at –20°C until assessments by radioimmunoassay (RIA) of T, LH, and CORT concentrations. Testes were removed for purification of Leydig cells. The animal protocol was approved by the Institutional Animal Care and Use Committee of The Rockefeller University.

Cell Isolation and Culture

Leydig cells were purified from rats as described previously (Klinefelter and Ewing, 1988). Leydig cell purity was assessed by histochemical staining for 3β-hydroxysteroid dehydrogenase activity, with 0.4 mM etiocholanolone as the steroid substrate (Payne et al, 1980). Consistently, greater than 95% of the Leydig cells were intensely stained.

For some studies, Leydig cells were cultured for 3 hours in buffered Dulbecco's Modified Eagle's Medium (DMEM):F12 culture medium alone, with a maximally stimulating dose of LH (100 ng/mL), or with substrate-saturating concentrations of 22(R)-hydroxycholesterol (2 ng/mL), pregnenolone (20 µM), progesterone (20 µM), or androstenedione (20 µM). The media were collected and stored at –20°C until the analysis of T by RIA.

RIA for Serum T, LH, and CORT Concentrations

Serum T concentrations were measured with a tritium-based RIA as previously described (Cochran et al, 1981). Serum LH concentrations were measured by RIA (Chandrashekar and Bartke, 1988). Rat LH standards NIDDK-r-LH-I9 and rat LH antibody NIDDK-anti-rLH-S-11 were obtained through the National Hormone and Pituitary Program. Rat 125I-labeled LH was obtained through Covance Laboratories (Vienna, Virginia). Immunoglobulin G antiserum was obtained from ICN Pharmaceuticals (Costa Mesa, California). Serum CORT was measured (Spencer et al, 1996). The CORT antiserum B3-163 was obtained from Endocrine Sciences (Calabasas, California). Interassay variations of the T, LH, and CORT RIAs were determined to be 7%–10% in each case.

Determination of 11βHSD Activities

Testes were removed from rats that had been treated in vivo with CORT and/or GA. The tunica was removed, testes were cut into slices, and 11βHSD activity was measured by incubating the slices with radiolabeled substrate as previously described (Ge et al, 1997). In brief, the testis slices (100 mg) were incubated with 1 µM [3H]CORT or [3H]11DHC in 0.5 mL phenol red–free medium (DMEM) at 34°C for 15 minutes. Media were harvested for measurement of substrate and product amounts, and the rates of 11βHSD activity were determined.

In vitro analyses of GA effects on testis 11βHSD1 oxidative and reductive activities were performed as previously described (Ge et al, 1997). In brief, first testicular microsomes were prepared (Ge et al, 1997). For analyses of oxidase or reductase activities, aliquots of 1 µg or 3 µg, respectively, were combined with 25 nM [3H]CORT or [3H]11DHC in 0.25 mL PBS buffer plus 400 µM NADP+ (for oxidase) or NADPH (for reductase) in the presence of increasing concentrations of GA. The incubations were for 60 minutes. 11βHSD assay reactions were stopped by adding 2 mL ice-cold ether. The steroids were extracted, and the organic layer was dried under nitrogen. The steroids were separated chromatographically on thin layer plates in chloroform:methanol (90:10), and radioactivity was measured with a scanning radiometer (System AR2000; Bioscan Inc, Washington, District of Columbia). The percentages of conversion of CORT to 11DHC and 11DHC to CORT were calculated by dividing the radioactive counts identified as 11DHC (or CORT, respectively) by the total counts associated with CORT plus 11DHC.

RT-PCR

Total RNA was isolated by a single-step method. Leydig cells purified after in vivo CORT and/or GA exposures were lysed with phenol and guanidium thiocyanate (Trireagent; Molecular Research Center, Cincinnati, Ohio) in accordance with the manufacturer's protocol. First-strand cDNA synthesis from 400 ng of total RNA was done using avian myeloblastosis virus reverse transcriptase, random primers, and deoxy-NTPs at 37°C for 75 minutes. The reaction was ended by heating at 95°C for 5 minutes. Target cDNA was coamplified with ribosomal protein S16 (Rps16) as the internal control in an aliquot of the synthesized product. Primers for the target cDNAs were synthesized on an oligonucleotide synthesizer (Gene Assembler Special; LKB, Rockville, Maryland) using published sequences. The sequences were 5'-AGGTGTAGCT CAGGACTTCA-3' (forward) and 5'-AGGAGGCTA TAAAGGACACC-3' (reverse) for Cyp11a1 (Oonk et al, 1989), 5'-TTGGGCATACTCAACAACCA-3' (forward) and 5'-ATGACACCGCTTTGCTCAG-3' (reverse) for StAR, and 5'-AAGTCTTCGGACGCAAGAAA-3' (forward) and 5'-TTGCCCAGAAGCAGAACAG-3' (reverse) for Rps16 (Chan et al, 1990). The expected product sizes were 399, 389, and 148 bp for Cyp11a1, StAR, and Rps16, respectively. PCR was initiated by the addition of Taq DNA polymerase, and continued for 35 cycles of 1 minute each at 94°C, 57°C, and 72°C. Preliminary studies showed that the cDNAs of interest were amplified linearly between 15 and 35 cycles of PCR. PCR products were separated on 3% agarose, visualized by ethidium bromide staining, and quantified on a densitometer (Kodak Scientific Imaging Systems, New York, New York) using 0.65 µg of 100-bp DNA ladder as standard. The mass of PCR products for Cyp11a1 and StAR was normalized to RPS16. The experiments were conducted 3 times, and RT-PCR assays were performed in triplicate.


Figure 1
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  		Figure 1. Serum corticosterone (CORT) levels after the administration of CORT, glycyrrhetinic acid (GA), or CORT + GA to rats. Values are means ± SEM (n x 17–18 rats). * indicates significant difference compared with control at P < .05.

 


Figure 2
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  		Figure 2. Suppression of 11βHSD1 oxidative and reductive activities in testis microsomes by glycyrrhetinic acid (GA). Radiolabeled substrates, either 25 nM corticosterone (CORT) or 11dehydroCORT (11DHC), were incubated with 1–3 µg microsomal protein with NADP+ or NADPH, respectively, in the presence of increasing concentrations of GA for 1 hour. IC50s were calculated.

 
Statistics

Each experiment was repeated 4 times. Serum T, LH, and CORT levels were analyzed by 1-way analysis of variance followed by Duncan's multiple comparison testing to identify significant differences between groups (SAS Institute, 2000). The IC50 for inhibition of 11βHSD1 by GA was calculated using nonlinear regression by Prism 3.0 (GraphPad Software, San Diego, California). The T data were analyzed by Student's t test, with a Sidak adjustment to the P value for planned comparisons to identify significant differences between treatment and control (SAS Institute, 2000). All data are expressed as means ± SEM. Differences were regarded as significant at P < .05.


   Results
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Discussion
 References
 
Effects of CORT and GA on Serum Levels of T, LH, and CORT

As seen in Figure 1, serum levels of CORT were significantly elevated after the administration to rats of CORT (twofold), GA (twofold), or GA + CORT (3.5-fold). GA inhibited 11βHSD1 oxidative activity more potently than its reductive activity; the IC50 for oxidative suppression was 6.6 nM, vs 255 nM for reductive inhibition (Figure 2). The 11βHSD oxidase activities in testis slices from rats treated with GA alone or CORT + GA were reduced to barely detectable levels when compared to control (Figure 3). Despite the elevation of serum CORT concentrations in response to CORT, GA, or CORT + GA, serum LH levels were not affected (Figure 4). Serum T levels were not affected by CORT or GA alone, but T was significantly reduced in the CORT + GA group (Figure 5).


Figure 3
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  		Figure 3. 11βHSD oxidative and reductive activities in testis after administration of corticosterone (CORT), glycyrrhetinic acid (GA), or CORT + GA to rats. Values are means ± SEM (n x 6 rat testes). * indicates a significant difference compared with control at P < .05.

 

Figure 4
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  		Figure 4. Serum luteinizing hormone (LH) levels after the administration of corticosterone (CORT), glycyrrhetinic acid (GA), or CORT + GA to rats. Values are means ± SEM (n x 17–18 rats).

 

Figure 5
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  		Figure 5. Serum testosterone (T) levels after the administration of corticosterone (CORT), glycyrrhetinic acid (GA), or CORT + GA to rats. Values are means ± SEM (n x 17–18 rats). * indicates a significant difference compared with control at P < .05.

 

Effects of CORT and GA Administration In Vivo on T Production by Purified Leydig Cells In Vitro

The capacity of Leydig cells to produce T in vitro was analyzed using cells isolated from rats that had been treated in vivo with CORT, GA, or CORT + GA. As shown in Figure 6, the capacities of Leydig cells to produce T under basal or LH-stimulated conditions were significantly reduced only after CORT + GA.


Figure 6
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  		Figure 6. Luteinizing hormone (LH)-stimulated testosterone (T) by Leydig cells isolated from rats that received corticosterone (CORT), glycyrrhetinic acid (GA), or CORT + GA. Values are means ± SEM (n x 12 different wells). * indicates a significant difference compared with control at P < .05.

 
The proteins encoded by StAR and Cyp11a1 are involved in the rate-limiting steps of cholesterol mobilization and side-chain cleavage, respectively, in the T biosynthetic pathway. The transcript levels of StAR and Cyp11a1 were analyzed, and both were significantly reduced only in the animals that received CORT + GA (Figure 7), indicating that GC-mediated suppression of T production could occur as early as the steps of cholesterol mobilization and side-chain cleavage. As seen in the Table, T production by Leydig cells isolated from rats that received CORT or GA alone was not reduced from control levels whether the cells were incubated with 22R-hydroxycholesterol, pregnenolone, progesterone, or androstenedione. In contrast, T production by cells isolated from rats that received CORT + GA were significantly reduced from control values when the cells were incubated with 22R-hydroxycholesterol, pregnenolone, or progesterone, but not with androstenedione.


Figure 7
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  		Figure 7. Messenger mRNA levels of Star and Cyp11a1 in Leydig cells isolated from rats after that received corticosterone (CORT), glycyrrhetinic acid (GA), or CORT + GA. RT-PCR was conducted with total RNA obtained from 4 separate experiments, and the audiogram shown is representative of the results obtained from the 4 experiments. Relative levels were compared to the internal control, ribosomal S16 protein (Rps16). Values are means ± SEM (n x 4 pools of Leydig cells). * indicates a significant difference compared with control at P < .05.

 


   Discussion
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 Results
 Discussion
References
 
The present study demonstrates that the administration to rats of GA and CORT in combination resulted in significant increases in serum CORT concentration, and significant reductions in serum T concentration and T production by Leydig cells isolated from the treated rats. Reduced T was correlated with reduced StAR and Cyp11A1 gene expression in Leydig cells. Serum levels of LH were unaffected by GA + CORT administration. Interestingly, although serum CORT concentrations were increased by the administration of GA or CORT alone, the elevations (about twofold) were insufficient to suppress T production. With GA + CORT, serum CORT levels increased to 3.5-fold (about 400 ng/mL), and this resulted in the inhibition of serum T and Leydig cell T production. These results suggest that CORT must reach a threshold level to effectively suppress T production.

Previous studies reported that GA administration inhibited 11βHSD in liver, kidney, and testis (Marandici and Monder, 1993), among other tissues. In the present study, GA, used at a relatively low dose (5 mg/kg) so as to minimize the inhibition of other hydroxysteroid dehydrogenases that are important in T biosynthesis (Armanini et al, 2003), inhibited Leydig cell 11βHSD1 oxidative activity more potently (IC50 6.6 nM) than reductive activity (IC50 255 nM), consistent with previous reports (Marandici and Monder, 1993). It is plausible that elevated local CORT levels following the administration of GA + CORT resulted from the inhibition of 11βHSD oxidase activity in Leydig cells, suppressing steroidogenesis as a direct effect on GC.Go


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  		Table. T production by Leydig cells isolated from rats after administration of CORT, GA, or CORT + GAa
 

The coadministration of CORT and GA in the present study increased serum CORT concentrations to approximately the levels attained under stress conditions (Hardy et al, 2005). The suppressive effects of stress on Leydig cell T production might be a consequence of direction action of GC on Leydig cells, but also might be attributable to a GC-mediated decrease in the release of LH from the pituitary and/or from altered neural input from sympathetic innervation (Lee et al, 2002). In the present study, serum LH levels were unchanged despite a 3.5-fold rise in serum CORT concentrations. Nitric oxide has been proposed as a possible stress-associated neurotransmitter (Kostic et al, 1998a; Herman and Rivier, 2006), but recent studies have failed to record changes in testicular nitrogen oxide synthase activity in relationship to declines in T production (Kostic et al, 2000; Weissman et al, 2007). These observations, taken together, are consistent with the hypothesis that stress-mediated increases in GC levels cause declines in T production by a direct action through Leydig cell GRs. This hypothesis, in turn, is consistent with observations of the response of Leydig cells to GCs in vitro (Monder et al, 1994b).

The magnitude of GC activity in the body is determined by the amount of the steroid in circulation, how much is bound to corticosteroid-binding globulin, and the degree of inactivation catalyzed by 11βHSD1 and 11βHSD2 oxidases. The 11βHSD2 isoform has a demonstrable role in preventing GC access to mineralocorticoid receptors, which might otherwise bind to this steroid, resulting in inappropriate cellular responses that mimic aldosterone action (Agarwal et al, 1994; Oppermann et al, 1997). is thought to potentiate GC action in tissues such as liver, where it stimulates glucose metabolism, and in lung, where it stimulates hyaline membrane maturation (Seckl and Walker, 2001). Both 11βHSD1 and 11βHSD2 are present in Leydig cells (Ge et al, 1997, 2005). It may be that the observed predominance of 11βHSD oxidative activity of Leydig cells in vitro requires the presence of 11βHSD2. Consistent with this hypothesis, antisense suppression of either isoform increases the sensitivity to CORT-mediated suppression of Leydig cell T production (Ge et al, 2005). In conclusion, excessive CORT levels by systematic inhibition of 11βHSD oxidase by GA in combination of administration of CORT might result in suppressed Leydig cell steroidogenic function.


   Footnotes
 
Supported by NIH grant HD-33000 (M.P.H.).

|| Deceased. Back


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