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11-Hydroxysteroid Dehydrogenase Type 1 Is Induced in Human Monocytes upon Differentiation to Macrophages¹

Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, NJ 07065

	Abstract

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11-hydroxysteroid dehydrogenases (11-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor , which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11-HSD1 activity by up to 10-fold. IFN-, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11-HSD1 and low levels of 11-HSD2. The expression of 11-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.

	Introduction

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The interconversion of pharmacologically active cortisol and inactive cortisone is accomplished by two independent 11-hydroxysteroid dehydrogenases (11-HSD)³ that exhibit tissue-specific expression (1). In intact cells, 11-HSD1 functions predominantly as a reductase, generating active cortisol from inactive cortisone and thereby enhancing activation of the glucocorticoid receptor. 11-HSD1 is broadly distributed among tissues, with predominant expression occurring in hepatic, adipose, gonadal, and central nervous system tissues. Mice with a targeted disruption of the 11-HSD1 gene are more resistant to hyperglycemia induced by stress or high-fat diet than their wild-type counterparts, consistent with the emerging notion that the activation of glucocorticoids by prereceptor metabolism may be central to the appearance of many sequelae of insulin resistance (2). 11-HSD2, which is mainly expressed in the placenta and aldosterone target tissues such as the kidney and colon, acts almost exclusively as a dehydrogenase, thereby preventing the activation of mineralocorticoid receptor-sensitive genes by excess cortisol (1). 18-Glycyrrhetinic acid, an active component of licorice, is an inhibitor of 11-HSD1 as well as 11-HSD2, and licorice ingestion or administration of 18-glycyrrhetinic acid or its hemisuccinate derivative carbenoxolone results in hypertension and metabolic alkalosis due to inhibition of 11-HSD2 (3, 4). Patients with mutations in the gene encoding 11-HSD2 suffer from the syndrome of "apparent mineralocorticoid excess" entailing hypokalemia and severe hypertension (5). Similar symptoms also were recently described for the 11-HSD2 knockout mice (2).

For several decades, synthetic glucocorticoids have found significant therapeutic use as anti-inflammatory agents in various diseases such as rheumatoid arthritis, allergic diseases, and bronchial asthma (6). Consistent with the pluripotent effects of glucocorticoids, the glucocorticoid receptor is widely distributed among peripheral tissues. In many instances, the tissue distribution of this receptor and that of 11-HSD1 are overlapping (1). Although glucocorticoids are commonly prescribed for their anti-inflammatory actions, to date relatively few studies address the involvement of 11-HSD in glucocorticoid-mediated immune functions. In one such study, the importance of prereceptor metabolism by 11-HSD enzymes in controlling inflammatory responses has been highlighted by demonstrating that pharmacological inhibition of 11-HSD activity present in skin lead to an augmentation of the anti-inflammatory action of topically applied cortisol on contact hypersensitivity responses (7).

We have now examined the expression of 11-HSD in a primary inflammatory effector cell, the monocyte/macrophage. Our studies confirm the complete absence of both 11-HSD1 and 11-HSD2 in freshly isolated circulating human monocytes. However, 11-reductase activity was induced during monocyte culture or after stimulation with the anti-inflammatory cytokines IL-4 and IL-13, strongly suggesting that it may play an important role in regulating the immune functions of these cells.

	Materials and Methods

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Materials

Cell culture medium was obtained from Life Technologies (Gaithersburg, MD). Recombinant cytokines were purchased from R&D Systems (Minneapolis, MN). FCS was obtained from HyClone Laboratories (Logan, UT). All other reagent grade chemicals were obtained from Sigma (St. Louis, MO). THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA). 1,25-Dihydroxyvitamin D₃ was obtained from Biomol (Plymouth Meeting, PA).

Purification of monocytes

Human PBMC for Figs. 1 and 2 were obtained by plasmapheresis (University of Pennsylvania, Philadelphia, PA). Removal of T lymphocytes and purification of monocytes was performed as described previously (8). Briefly, the cells were washed with RPMI 1640 medium and further purified by centrifugation by lymphocyte separation medium (ICN, Aurora, OH). The interphase containing mononuclear cells was harvested. T lymphocytes were removed by the SRBC rosetting method (9). For all other experiments, monocytes were prepared from heparinized whole blood from healthy donors by lymphocyte separation medium as described above without further removal of T lymphocytes. Both methods produced essentially the same results.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Expression of 11-HSD and reductase activities in human whole blood and peripheral blood monocytes during differentiation. A, Heparinized human whole blood, diluted to 50% with RPMI 1640 medium, was transferred to individual wells of a 24-well dish and incubated for 24 h with 15 nM [3H]cortisol (for dehydrogenase activity measurements) or [3H]cortisone (for reductase activity) in the absence and presence of 50 ng/ml IL-4. Corticosteroids were extracted with ethyl acetate and further separated by reversed phase HPLC. Activities were determined by measuring the conversion (%) of the tritiated substrate to cortisone or cortisol, respectively, as indicated. B, Freshly isolated human peripheral blood monocytes were transferred to Teflon beakers. At the indicated day of incubation, aliquots of the cells (containing 1 x 106 cells) was transferred to individual wells of a 12-well cell culture dish, and 15 nM [3H]cortisol or [3H]cortisone was added to the medium. The cell medium was harvested after 24 h of incubation. The experiment is a representative of two similar experiments conducted.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Expression of 11-HSD1 and 11-HSD2 mRNA in human peripheral blood monocytes. Aliquots of the cells (1 x 106) from Fig. 1B were removed from the Teflon dishes at the indicated day of culture and lysed in TRIzol reagent followed by isolation of total RNA. A, RT-PCR of 11-HSD1. Total RNA from monocytes was harvested on day 0, 2, 4, or 7 (as indicated in the template line) and subjected to DNase treatment to remove possible contamination with genomic DNA. After reverse-transcription in the presence (+) or absence (-) of reverse transcriptase (RT), PCR was performed with primer pairs specific for human 11-HSD1 or 11-HSD2. PPAR2 served as a differentiation marker; GAPDH was used as an internal control to assess the relative effectiveness of the RT-PCR. Plasmids containing the corresponding cDNAs for 11-HSD1 (H1), 11-HSD2 (H2), and PPAR2 (P) served as PCR controls. B, Real-time quantitative (TaqMan) PCR of 11-HSD1. Real-time quantitative PCR was performed with total RNA (isolated and processed as above) primers and a probe specific for human 11-HSD1. The amount of 11-HSD1 mRNA was calculated relative to the amount of GAPDH mRNA in the samples, and refers to the fold induction compared with the amount of 11-HSD1 detected on Day 0.

Culture of mononuclear phagocytes in suspension by incubation on a Teflon surface to which cells do not adhere has been described earlier (8, 10). Briefly, 1 x 10⁷ monocytes were resuspended in 10 ml of RPMI 1640 medium with L-glutamine and 14% normal human serum per 60-ml Teflon beaker. The loosely capped beakers were incubated at 37°C in a 5% CO₂ atmosphere. Cell recovery from each beaker was 90%.

RNA isolation and analysis

Total RNA was isolated with TRIzol reagent (Life Technologies) and was further treated with DNase I to remove potential contamination by genomic DNA, followed by reverse transcription as described previously (8). Kit-provided human placental RNA (1 µg) was used as a control. Aliquots were subjected to PCR amplification with Taq polymerase (Fisher, Pittsburgh, PA). The following primers were used: human G3PDH amplimer set 5406 (Clontech Laboratories, Palo Alto, CA); human peroxisome proliferator activating receptor (PPAR) forward primer, 5'-GGAAAGACAACAGACAAATCAC; human PPAR reverse primer, 5'-TGCATTGAACTTCACAGCAAAC; human 11-HSD1 forward primer, 5'-TGCTCATTCTCAACCACATCAC; human 11-HSD1 reverse primer, 5'-ACAGAACAGTCCCAAAATCCC; human 11-HSD2 forward primer, 5'-GGCTGTGACTCTGGTTTTG; human 11-HSD2 reverse primer, 5'-AACTGCCCATGCAAGTGCTC.

Expression levels of specific mRNAs were quantitated by quantitative fluorescent real-time PCR. RNA was first reverse transcribed, and amplification of each target cDNA then was performed with TaqMan PCR reagent kits in the ABI Prism 7700 sequence detection system according to the protocols provided by the manufacturer (Applied Biosystems, Branchburg, NJ). The following primer/probe sets were used for the amplification step: human 11-HSD1 forward primer (5'-AAGCAGAGCAATGGCAGCAT); human 11-HSD1 reverse primer (5'-GAGCAATCATAGGCTGGGTCAT); human 11-HSD1 probe (5'-CGTCATCTCCTCCTTGGCTGGGAA); human 11-HSD2 forward primer, (5'-GAGACATTAGCCGCTTGCTAGAG); human 11-HSD2 reverse primer (5'-GTTGACGGGCCCCACAG); human 11-HSD2 probe, (5'-CCAAGGCCCACACCACCAGCA). The TaqMan probes described above consist of an oligonucleotide with a 5'-end reporter dye (FAM, 6-carboxy-fluorescein) and a 3'-end quencher dye (TAMRA, 6-carboxy-tetramethyl-rhodamine). These primer/probe sets span intron/exon junctions to minimize amplification of contaminating genomic DNA in our RNA samples. The levels of mRNA were normalized to the amount of GAPDH RNA (primers and probes commercially available from Applied Biosystems) or the 23-kDa highly basic protein RNA (forward primer, 5'-GCTGGAAGTACCAGGCAGTGA; reverse primer 5'-CCGGTAGTGGATCTTGGCTTT; probe, 5'-TCTTTCCTCTTCTCCTCCAGGGTGGCT) detected in each sample. No significant signal was obtained in control PCR performed with samples obtained from reverse-transcription reactions conducted in the absence of reverse transcriptase and the primer/probe sets presented above (data not shown).

Assay of 11-HSD activity

11-HSD activity was determined in intact cells cultured as described above by measuring the interconversion of [³H]cortisone to [³H]cortisol. After treatment of the cells as described in the figure legends, 15 nM [³H]cortisone or [³H]cortisol was added to the medium (sp. act., 50 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO). After the appropriate incubation times, steroids were extracted with 3 volumes of ethyl acetate. The organic phase was collected, evaporated to dryness, and reconstituted in DMSO containing 16 µg/ml each of unlabeled cortisone and cortisol. The samples were injected into a Waters HPLC system with an Inertsil 5-µm ODS2 column (Metachem Technologies, Torrence, CA) and eluted with gradient of 70% solvent A (water-methanol-trifluoroacetic acid 90:10:0.05 v/v/v)/30% solvent B (water-methanol-trifluoroacetic acid 10:90:0.05 v/v/v) to 40% solvent A/60% solvent B. Eluted tritiated steroids were detected with a -RAM flow-through radioisotope detector (IN/US Systems, Tampa, FL). The conversion of [³H]cortisone or [³H]cortisol to their corresponding tritiated products was calculated as an index of activity.

	Results

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11-HSD1 is expressed in cultured human monocytes

To investigate whether 11-HSD or reductase activity is present in circulating immune cells, we added [³H]cortisone or [³H]cortisol to heparinized whole human blood. We were not able to detect any enzymatic conversion of the substrates in whole blood, suggesting that none of the cells present in whole blood, including lymphoid cells, mononuclear phagocytes, and polymorphonuclear granulocytes, contain significant levels of 11-HSD1 or 11-HSD2 (Fig. 1A, no addition). We further confirmed the complete absence of 11-HSD or reductase activity in isolated human PBMC (Fig. 1B, day 0). However, during cultivation of the monocytes, an increase in reductase activity, determined by the appearance of [³H]cortisol in the medium, was observed upon differentiation into macrophages (Fig. 1B). This up-regulation of enzyme activity was observed regardless of whether the monocytes were maintained in suspension culture in Teflon beakers (Fig. 1B) or as an adherent monocyte layer in plastic tissue culture dishes (data not shown and see Fig. 4A). 11-Dehydrogenase activity was almost completely absent from the cells, as evidenced by the lack of conversion of [³H]cortisol into [³H]cortisone (or any other [³H]-labeled metabolites). Essentially, all radioactivity in the HPLC chromatograms was found in the position of cortisol and cortisone, suggesting that the cells did not convert either [³H]cortisone or [³H]cortisol into other metabolites (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Time (A) and dose response (B) of IL-4- and IL-13-mediated induction of 11-HSD1. Freshly isolated human monocytes were distributed to 24-well dishes. Recombinant IL-4 or IL-13 was added to the culture at the indicated concentrations and the cells were incubated for 24 h (B) or as indicated with 50 ng/ml IL-4 (A). [3H]Cortisone (15 nM) was added to the medium, and corticosteroids were extracted after further incubation for 24 h and analyzed as described in Fig. 1. The experiment was repeated once with essentially the same results.

To rule out the possibility that the reductase activity found in the differentiated monocytes represented a novel, as yet undescribed, form of 11-HSD we PCR-amplified cDNA encompassing the entire open reading frame from these cells. The resulting DNA sequence showed complete identity to the published sequence of 11-HSD1 (data not shown and Ref. 12). In summary, these findings strongly suggest that the observed reductase activity is attributable to the known 11-HSD1.

Monocyte expression of 11-HSD1 is induced by IL-4/IL-13 and 1,25-dihydroxyvitamin D₃

We next sought whether the expression of 11-HSD1 could be regulated by pro- or anti-inflammatory agents. Fresh monocytes were exposed to a panel of known effectors of immune function such as cytokines, LPS, and differentiating agents. TNF- and IL-1 had no effect on 11-HSD1 expression, and the apparent small suppressive effects of PMA and LPS on 11-HSD1 shown in Fig. 3 were not reliably reproducible and, therefore, insignificant (data not shown). In separate studies, treatment of cells with LPS, PMA, TNF-, or IL-1 resulted in the release of proinflammatory mediators, confirming the biological activity of these agents (data not shown). In contrast, the Th2 cytokines IL-4 and IL-13 and, to a lesser degree, the differentiating agent 1,25-dihydroxyvitamin D₃, were able to significantly and reproducibly induce the 11-HSD1 activity. The increase in 11-reductase activity with IL-4 and IL-13 occurred in a time- and dose-dependent manner (Fig. 4) and at concentrations of IL-4 that completely abolished IL-1 expression in response to LPS stimulation of the monocytes (data not shown). As with purified monocytes, incubation of human whole blood with IL-4 was able to induce the expression of 11-hydroxysteroid reductase activity (Fig. 1A).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Regulation of 11-HSD1 activity in human peripheral blood monocytes. Freshly prepared human PBMC were incubated with the indicated reagents: IL-4 (50 ng/ml), IL-13 (50 ng/ml), TNF- (10 ng/ml), IL-1 (10 ng/ml), 18-glycyrrhetinic acid (18-GA, 20 µM), PMA (100 ng/ml) 1,25-dihydroxyvitamin D3 (500 ng/ml), or LPS (100 ng/ml). Wells containing medium but no cells served as an additional control. After 24 h, 15 nM [3H]cortisol or [3H]cortisone was added to the medium to determine 11-HSD reductase activity. After an additional 24 h of incubation, the medium was harvested and processed for determination of the resulting corticosteroids as described in Fig. 1. The experiment is a representative of four similar experiments.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. IFN- antagonizes the IL-4- or IL-13-mediated induction of 11-HSD1. Human PBMC were distributed to 24-well dishes (3.5 x 105 cells per well). Recombinant IL-4 (50 ng/ml), IL-13 (50 ng/ml), and/or IFN- (100 ng/ml) was added to the medium as indicated. A, After 24 h, [3H]cortisone (15 nM) was added to the medium, and corticosteroids were extracted after further incubation for 24 h and analyzed as described in Fig. 2. The experiment is a representative of three similar experiments. B, After a total of 48 h of incubation, total RNA was isolated by the TRIzol lysis method and further processed for gene expression by real-time quantitative PCR as outlined in Fig. 2. Quantitation of 11-HSD1 gene expression was performed relative to the expression of the 23-kDa highly basic protein.

Expression of 11-HSD1 in macrophage cell lines

Screening of the monocyte/macrophage-like cell lines U937, RAW 264.7, and THP-1 revealed that only the latter cell line showed significant, albeit low, 11-hydroxysteroid reductase activity, whereas dehydrogenase activity was completely absent from all cell lines (Fig. 6 and data not shown). The expression of 11-HSD1 in THP-1 cells was confirmed by quantitative real-time PCR analysis. As shown in Fig. 6, stimulation of THP-1 cells with LPS increased the expression of 11-HSD1 4-fold.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. 11-HSD1 in THP-1 cells is up-regulated by LPS. THP-1 cells were distributed to six-well dishes (5 x 105 cells per well) in RPMI 1640 medium containing 0.25% normal human plasma. LPS (3 ng/ml) was added to the cells, and the cells were further incubated for 16 h. Real-time quantitative PCR was performed with total RNA and primers and a probe specific for human 11-HSD1. The amount of 11-HSD1 mRNA was calculated relative to the amount of GAPDH mRNA in the untreated sample, and refers to the fold induction compared with the amount of 11-HSD1 RNA detected in control THP1 cells that were not treated with LPS. The experiment is a representative of three similar experiments.

	Discussion

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View larger version (55K): [in this window] [in a new window]   FIGURE 7. Model for the anti-inflammatory action of IL-4 mediated by 11-HSD1. IL-4 induces the expression of 11-HSD1 in monocytes. The increase in enzymatic activity raises the intracellular levels of the active glucocorticoid, cortisol. Cortisol is known to transrepress the expression of proinflammatory mediators during an inflammatory insult. The effects of IL-4 on 11-HSD1 expression are antagonized by IFN-.

We discovered that the expression of 11-HSD1 is greatly up-regulated during differentiation of monocytes into macrophages. Furthermore, incubation of monocytes with the well known differentiating agent 1,25-dihydroxyvitamin-D₃ also induced 11-hydroxysteroid reductase activity. This observation suggests that, during differentiation into macrophages, monocytes will be exposed to greater levels of cortisol through the action of 11-HSD1 and will thereby exhibit a blunting of inflammatory potency (Fig. 7).

None of the proinflammatory stimuli investigated, TNF-, IL-1, LPS (Fig. 3), or IL-6 (data not shown), increased the 11-HSD1 activity in the monocytes. This is in keeping with the potential anti-inflammatory role of 11-HSD1 proposed above. In contrast with the present studies in monocytes, we have recently observed that TNF- and IL-1 were potent inducers of 11-HSD1 in human aortic and bronchial smooth muscle cells (20). Moreover, Escher et al. (21) have shown that 11-HSD1 is expressed in rat glomerular mesangial cells, where it is up-regulated by TNF- and IL-1. These data suggest that 11-HSD1 may play an important role in regulating inflammatory responses not only in monocytes but also in the artery wall, lung, and kidney, and that changes in 11-HSD1 expression may be tissue-selective.

We further observed that 11-HSD1 in monocytes is strongly up-regulated by the Th2 cytokines IL-4 and IL-13. These cytokines cause the differentiation of immature T cells into Th2 cells, which in turn are the main producers of IL-4 and IL-13. Both cytokines are potent suppressors of LPS-inducible inflammatory mediator production (22). The activities of IL-4 and IL-13 are very similar because the predominant signaling chain, IL-4R, of the IL-4R complex is common to both cytokines (23, 24). IL-4 and IL-13 responses are mainly mediated by activation of the latent cytoplasmic transcription factor STAT6 (25). Interestingly, several STAT motifs are present in the presumed promoter region of human 11-HSD1 (J. Yuan and R. Thieringer, unpublished observation). In keeping with the suppressive function of IL-4 and IL-13, the up-regulation of 11-HSD1 is predicted to raise the level of active intracellular cortisol and further suppress the production of inflammatory mediators by macrophages (Fig. 7). Induction of 11-HSD1 may thus provide a novel additional mechanism for the known anti-inflammatory activities of these cytokines.

IFN-, which is expressed primarily by Th1 cells, is a known antagonist of IL-4 and IL-13 (13), and, accordingly, effectively suppressed the induction of 11-HSD1 activity in monocytes by these Th2 cytokines. IFN- is known to enhance the responsiveness of macrophages to LPS (26, 27, 28). Consistent with this stimulatory role, the suppression of 11-HSD1 activity by IFN- would be expected to lower the level of active intracellular cortisol and further enable the production of inflammatory mediators. This effect may be even further enhanced in vivo, because glucocorticoids increase the expression of IL-4 from Th2 cells and suppress the secretion of IFN- from Th1 cells (19).

Interestingly, the combination of IL-4 and IL-2 has been shown to reduce the binding affinity of glucocorticoid receptor in T lymphocytes and to blunt cellular responses to glucocorticoids (29). These effects could be blocked by coincubation with IFN-. Also, Spahn et al. (30) have reported that IL-13 induces diminished glucocorticoid receptor binding affinity in monocytes and reduces the suppression of LPS-induced cytokine release by glucocorticoids. Thus, it becomes increasingly apparent that the production of cytokines as well as the cellular responsiveness to glucocorticoids is intricately regulated by a multitude of mechanisms. These include direct effects on receptor function as well as the modulation of ligand availability via prereceptor metabolism and further involve the cross-talk between different cell types such as Th1 and Th2 cells and monocyte/macrophages. Recent studies from our laboratory have described the regulation of 11-HSD1 by nuclear receptors known as PPAR. These receptors bind fatty acids and prostanoids and are thought to regulate lipid metabolism and inflammatory events. We observed that in liver, PPAR- agonists down-regulate 11-HSD1 expression (31), and PPAR agonists reduce the expression of 11-HSD1 in adipocytes (32). Preliminary results indicate that, unlike with hepatic tissue or adipocytes, the expression of 11-HSD1 in macrophages is not affected by PPAR agonists (data not shown). These findings provide a second example of the tissue selectivity of the regulation of 11-HSD1.

Glass and coworkers (33) have shown previously that IL-4 induces the expression of a 12/15-lipoxygenase, which generates 13-HODE and 15-HETE, potential endogenous ligands for PPAR, from linoleic and arachidonic acids. IL-4 also induces PPAR expression, and it was proposed that the suppression of nitric oxide production by IL-4 may be explained, in part, by the coordinate induction of PPAR and the production of its activating ligands by 15-lipoxygenase (33). Because cortisol also suppresses nitric oxide synthase expression (34), the work described here suggests an additional pathway for IL-4 to exert its anti-inflammatory effects. Studies in mice deficient in 11-HSD1 will be extremely instructive in determining the contribution of this pathway.

	Acknowledgments

 
We thank Steven Mundt and Shu-Yin Ho for important technical contributions to this work. We also thank Dr. Jeffrey Yuan for advice on gene analysis.

	Footnotes

 
¹ Parts of this study were previously presented at The Endocrine Society’s 82nd Meeting, June 21–24, 2000, Toronto, Ontario, Canada (Abstr. 837).

² Address correspondence and reprint requests to Dr. Rolf Thieringer, Merck Research Laboratories, 126 East Lincoln Avenue, RY80-B11, Rahway, NJ 07065. E-mail address: rolf_thieringer{at}merck.com

³ Abbreviations used in this paper: 11-HSD, 11-hydroxysteroid dehydrogenases; PPAR, peroxisome proliferator activating receptor.

Received for publication November 13, 2000. Accepted for publication April 23, 2001.

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