The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

Differential expression of placental 11β-hydroxysteroid dehydrogenases in pregnant women with diet-treated gestational diabetes mellitus

Fetal exposure to excess glucocorticoid is one of the critical factors for the fetal origins of adult diseases. However, the mechanism of the local regulation of glucocorticoid activity in the human placenta of pregnancies complicated with gestational diabetes mellitus (GDM) has not been fully understood. We investigated placental 11β-hydroxysteroid dehydrogenases (11β-HSDs) expression, and analyzed their relationship with cortisol levels in maternal and umbilical vein. Pregnant women with GDM after diet intervention (n=23) or normal glucose tolerance (NGT, n=22) were studied at the community-based hospital. We collected maternal and umbilical venous cord blood and placental tissues from both groups. Explanted placentas from NGT were cultured with palmitic acid, dexamethasone, insulin or their mixture for 24-h. We examined plasma cortisol, cortisone to cortisol ratio, insulin, the homeostasis model assessment of insulin resistance index (HOMA-IR) and the insulin secretion index. Quantitative real-time PCR, Western blot and immunohistochemical assay were applied for the measurement of 11β-HSD1 and 11β-HSD2 mRNA and protein. GDM had higher maternal cortisol levels, HOMA-IR, insulin secretion index and higher cortisone to cortisol ratio in umbilical vein. No significant change in cortisol levels in umbilical vein and newborn weight was found. GDM placental 11β-HSD1 levels decreased while 11β-HSD2 increased. Treatment of placenta explants from NGT with palmitic acid, dexamethasone, insulin or their combination resulted in a significant drop of 11β-HSD1 and increase in 11β-HSD2. Differential expression of 11β-HSDs in diet-treated GDM placenta provides a protective mechanism for the fetus throughout the adverse environment of pregnancy by limiting excessive exposure of the fetus to glucocorticoid.     

Design,synthesis, and biological evaluation of novel selective peptide inhibitors of 11β-hydroxysteroid dehydrogenase 1

The enzyme 11β-HSD1 plays a crucial role in the tissue-specific regulation of cortisol levels and it has been associated with various diseases. Inhibition of 11β-HSD1 is an attractive intervention strategy and the discovery of novel selective 11β-HSD1 inhibitors is of high relevance. In this study, we identified and evaluated a new series of selective peptide 11β-HSD1 inhibitors with potential for skin care applications. This novel scaffold was designed with the aid of molecular modeling and two previously reported inhibitors. SAR optimization yielded highly active peptides (IC₅₀ below 400?nM) that were inactive at 1?µM concentration against structurally related enzymes (11β-HSD2, 17β-HSD1 and 17β-HSD2). The best performing peptides inhibited the conversion of cortisone into cortisol in primary human keratinocytes and the most active compound, 5d, was further shown to reverse cortisone-induced collagen damage in human ex-vivo tissue.     

IL-13-induced changes in endogenous glucocorticoid metabolism in the lung regulate the proasthmatic response

Endogenous glucocorticoid (GC) activation is regulated by the intracellular GC-activating and -inactivating enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)1 and 11β-HSD2, respectively, that catalyze interconversion of inert cortisone and its bioactive metabolite cortisol. Because endogenous GCs are critically implicated in suppressing the asthmatic state, this study examined the roles of the 11β-HSD enzymes in regulating GC activation and bronchoprotection during proasthmatic stimulation. Airway hyperresponsiveness to methacholine and inflammation were assessed in rabbits following inhalation of the proasthmatic/proinflammatory cytokine IL-13 with and without pretreatment with the 11β-HSD inhibitor carbenoxolone (CBX). Additionally, IL-13-induced changes in 11β-HSD isozyme expression and GC metabolism were examined in epithelium-intact and -denuded tracheal segments and peripheral lung tissues. Finally, the effects of pretreatment with CBX or 11β-HSD2-targeted siRNAs were investigated with respect to cortisol prevention of IL-13-induced airway constrictor hyperresponsiveness and eotaxin-3 production by airway epithelial cells. IL-13-exposed rabbits exhibited airway hyperresponsiveness, inflammation, and elevated bronchoalveolar lung fluid levels of eotaxin-3. These responses were inhibited by pretreatment with CBX, suggesting a permissive proasthmatic role for 11β-HSD2. Supporting this concept, extended studies demonstrated that 1) IL-13-treated tracheal epithelium and peripheral lung tissues exhibit upregulated 11β-HSD2 activity, 2) the latter impairs cortisone-induced cortisol accumulation and the ability of administered cortisol to prevent both IL-13-induced heightened airway contractility and eotaxin-3 release from epithelial cells, and 3) these proasthmatic responses are prevented by cortisol administration in the presence of 11β-HSD2 inhibition. Collectively, these data demonstrate that the proasthmatic effects of IL-13 are enabled by impaired endogenous GC activation in the lung that is attributed to upregulation of 11β-HSD2 in the pulmonary epithelium.     

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/β ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)-1 (cortisone to cortisol conversion), 11β-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11β-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.     

Differential Regulation of 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Patients with Differing Etiologies of Hypopituitarism

Objective: Pituitary patients with different etiologies of hypopituitarism exhibit differing phenotypes, despite similar replacement therapy strategies. We hypothesized that differential regulation of the isoenzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which mediates the net autocrine conversion of cortisone to cortisol in adipose tissues and liver, may play a role.Methods: We studied 11β-HSD1 activity (using urine cortisol/cortisone metabolites ratio) in 36 hypopituitary patients with treated craniopharyngiomas, treated remitted Cushing disease, and treated nonfunctioning pituitary adenomas + prolactinomas on and off growth hormone (GH) replacement.Results: 11β-HSD1 activity was higher in subjects with craniopharyngioma both on and off GH, as evidenced by increased tetrahydrocortisol to tetrahydrocortisone metabolite ratios compared to other diagnostic groups, but there was no difference in body mass index, insulin levels, serum hormone measurements, or hydrocortisone dose between groups.Conclusion: Craniopharyngiomas are associated with enhanced 11β-HSD1 activity compared to other diagnostic hypopituitary groups, and this may contribute to the adverse phenotypic and metabolic features seen in this condition.Abbreviations: BMI = body mass index; Em = cortisone metabolites; Fm = cortisol metabolites; GH = growth hormone; 11β-HSD1 = 11β-hydroxysteroid dehydrogenase type 1; IGF-1 = insulin-like growth factor 1; NFPA = nonfunctioning pituitary adenoma; THE = tetrahydrocortisone; THF = tetrahydrocortisol     

Evolution of 11β-hydroxysteroid dehydrogenase-type 1 and 11β-hydroxysteroid dehydrogenase-type 3

A key regulator of glucocorticoid action is 11β-hydroxysteroid dehydrogenase-type 1 (11β-HSD1), which catalyzes the conversion of cortisone to cortisol, the biologically active glucocorticoid. 11β-HSD1 is a paralog of 11β-HSD3, whose physiological function remains unclear. As reported here, 11β-HSD3 has orthologs in sea urchin, amphioxus and Ciona, while 11β-HSD1 first appears in sharks. Thus, 11β-HSD3 arose before the evolution of glucocorticoid signaling, suggesting different ancestral function(s) for 11β-HSD3. Four perplexing findings arise from this evolutionary analysis: (1) 11β-HSD1 is not present in a ray-finned fish genome, (2) zebrafish and fathead minnow contain two isoforms of 11β-HSD3; (3) neither rat nor mouse contain 11β-HSD3 and (4) amphioxus contains 16 11β-HSD3 paralogs.     

Dynamics of endogenous glucocorticoid secretion and its metabolism in Kawasaki disease

Objective

Kawasaki disease (KD) is a severe inflammatory disease that occurs in childhood. Recently, the initial corticosteroid therapy for KD has been reconsidered because its efficacy is controversial. The aim of this study was to evaluate the dynamic change in endogenous glucocorticoid levels and their relation with 11beta-hydroxysteroid dehydrogenase (11β-HSD) activity in the acute phase of KD.

Study design

Sixteen KD patients were investigated. Cortisol and cortisone levels, the cortisol/cortisone ratio and C-reactive protein (CRP) levels were measured on admission, before the first intravenous immunoglobulin (IVIG) therapy and convalescence.

Results

The 16 patients were divided into two groups. Group A included patients who received the first IVIG on admission and blood samples were collected before the first IVIG and convalescence. Group B included patients whose blood samples were collected at three different time points (on admission, before the first IVIG, and convalescence). CRP and cortisol levels and the cortisol/cortisol ratio were markedly higher before the first IVIG than those of convalescence in all patients except in one patient. In Group B patients, both serum cortisol levels and the cortisol/cortisone ratio on admission were significantly increased compared with those before the first IVIG (cortisol: p < 0.005, cortisol/cortisone: p < 0.001).

Conclusions

Decreases in cortisol levels and the cortisol/cortisone ratio before the first IVIG may be explained by a reduction in adrenal secretion and/or local glucocorticoid action through 11β-HSD activity. These findings suggest that exogenous glucocorticoid treatment in combination with the first IVIG at the acute stage may play a synergetic role in KD.     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10⁻¹³ M cortisol, whereas 1 × 10⁻⁵ M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.     

The effect of glucose on the metabolism and excretion of cortisol in man

The 24 hour urinary free cortisol and cortisone excretion after an oral 100 g glucose load was measured in 60 males (aged 22-56) divided into three groups. G-I consisted of 10 healthy men, G-II of 37 surgical patients and G-III comprised 23 patients with atherosclerotic peripheral vascular disease. The followed subjects responded to the glucose ingestion accordingly to their cortisol excretion. Subjects with an urinary cortisol excretion up to 200 micrograms/24 h responded to the glucose load with an increase of excretion in free cortisol and cortisone. Subjects with the excretion of cortisol above 200 micrograms/24 h responded unambiguously with a decrease in their excretion. We suggest that these changes in both directions can be explained by the available amount of NADPH in the liver. In patients with atherosclerotic peripheral vascular disease, in whom disturbances in lipid and carbohydrate metabolism can be proposed, the response of free corticoids, namely the respond of cortisone, are unequal.     

Regulation of lipogenesis by glucocorticoids and insulin in human adipose tissue

Patients with glucocorticoid (GC) excess, Cushing's syndrome, develop a classic phenotype characterized by central obesity and insulin resistance. GCs are known to increase the release of fatty acids from adipose, by stimulating lipolysis, however, the impact of GCs on the processes that regulate lipid accumulation has not been explored. Intracellular levels of active GC are dependent upon the activity of 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) and we have hypothesized that 11β-HSD1 activity can regulate lipid homeostasis in human adipose tissue (Chub-S7 cell line and primary cultures of human subcutaneous (sc) and omental (om) adipocytes. Across adipocyte differentiation, lipogenesis increased whilst β-oxidation decreased. GC treatment decreased lipogenesis but did not alter rates of β-oxidation in Chub-S7 cells, whilst insulin increased lipogenesis in all adipocyte cell models. Low dose Dexamethasone pre-treatment (5 nM) of Chub-S7 cells augmented the ability of insulin to stimulate lipogenesis and there was no evidence of adipose tissue insulin resistance in primary sc cells. Both cortisol and cortisone decreased lipogenesis; selective 11β-HSD1 inhibition completely abolished cortisone-mediated repression of lipogenesis. GCs have potent actions upon lipid homeostasis and these effects are dependent upon interactions with insulin. These in vitro data suggest that manipulation of GC availability through selective 11β-HSD1 inhibition modifies lipid homeostasis in human adipocytes.     

Glucocorticoid Metabolism in Hypertensive Disorders of Pregnancy: Analysis of Plasma and Urinary Cortisol and Cortisone

Objectives

The aim of the study was to analyze the plasma and urinary cortisol (F) and cortisone (E) levels in normotensive and hypertensive pregnant women. The parameters known to reflect the function of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were calculated to verify the changes in glucocorticoid balance over the course of gestational hypertension (GH) and pre-eclampsia (PE).

Materials and Methods

This retrospective case-control study included women in the third trimester of pregnancy, diagnosed with: GH (n = 29), PE (n = 26), or chronic hypertension (CH; n = 22). Normotensive women in their third trimester of pregnancy were also included (controls; n = 43). The plasma and urinary F and E levels were measured with the HPLC-FLD method. The 11β-HSD2 function was estimated by calculating the following ratios: plasma F/E and urinary free F to urinary free E (UFF/UFE). A statistical analysis was performed based on case-control structure.

Results and Discussion

PE was characterized by lower plasma F levels (639.0 nmol/L), UFF/Cr levels (3.80 μg/mmol) and F/E ratio (3.46) compared with that of the controls (811.7 nmol/L, 6.28 μg/mmol and 5.19, respectively) with marked abnormalities observed in the changes of F/E and UFF/UFE ratios with advancing gestation. GH patients showed significant disparities in the urinary steroid profile with lower UFF/UFE ratio (0.330 vs. 0.401) compared with the normotensive controls and abnormal changes in the UFF/UFE throughout pregnancy. The observed tendency towards lower F/E and UFF/UFE ratios in PE and GH patients may reflect more intensive F metabolism over the course of those disorders. In the normal pregnancy group, the plasma F/E and UFF/UFE ratios tended to present inverse correlations with advancing gestation. This trend was much less marked in PE and GH patients, suggesting that the abnormalities in 11β-HSD2 functions progressed with the GA. The birth weights of neonates born from pre-eclamptic pregnancies were lower than those from uncomplicated pregnancies, although only when the babies were born prematurely. Children born at term to normotensive mothers or mothers suffering from PE had comparable birth weights.     

Reciprocal regulation of 11��-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression by dexamethasone inhibits human coronary artery smooth muscle cell proliferation in vitro

The actions of glucocorticoids are mediated, in part, by 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), which amplifies their effects at the pre-receptor level by converting cortisone to cortisol. Glucocorticoids, such as dexamethasone, inhibit vascular smooth muscle cell proliferation; however, the role of 11β-HSD1 in this response remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with dexamethasone (10(-9)-10(-6) mol/l) and found that after 72?h dexamethasone increased 11β-HSD1 expression (14.16?±?1.6-fold, P?相似文献   

Type 2 11β-hydroxysteroid dehydrogenase in foetal and adult life

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyse the interconversion of active cortisol to inactive cortisone; 11β-HSD1 is a low affinity, NADP(H)-dependent dehydrogenase/oxo-reductase, and 11β-HSD2 a high affinity, NAD-dependent dehydrogenase. Because of the importance of 11β-HSD in regulating corticosteroid hormone action, we have analysed the distribution of the 11β-HSD isoforms in human adult and foetal tissues (including placenta), and, in addition have performed a series of substrate specificity studies on the novel, kidney 11β-HSD2 isoform. Using an RT-PCR approach, we failed to detect 11β-HSD1 mRNA in any human mid-gestational foetal tissues. In contrast 11β-HSD2 mRNA was present in foetal lung, adrenal, colon and kidney. In adult tissues 11β-HSD2 gene expression was confined to the mineralocorticoid target tissues, kidney and colon, whilst 11β-HSD1 was expressed predominantly in glucocorticoid target tissues, liver, lung, pituitary and cerebellum. In human kidney homogenates, 11-hydroxylated progesterone derivatives, glycyrrhetinic acid, corticosterone and the “end products” cortisone and 11-dehydrocorticosterone were potent inhibitors of the NAD-dependent conversion of cortisol to cortisone. Finally high levels of 11β-HSD2 mRNA and activity were observed in term placentae, which correlated positively with foetal weight. The tissue-specific distribution of the 11β-HSD isoforms is in keeping with their differential roles, 11β-HSD1 regulating glucocorticoid hormone action and 11β-HSD2 mineralocorticoid hormone action. The correlation of 11β-HSD2 activity in the placenta with foetal weight suggests, in addition, a crucial role for this enzyme in foetal development, possibly in mediating ontogeny of the foetal hypothalamo-pituitary-adrenal axis.     

The development and SAR of pyrrolidine carboxamide 11β-HSD1 inhibitors

The design and development of a series of highly selective pyrrolidine carboxamide 11β-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11β-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11β-HSD1 selective inhibitor 42.     

In vivo activity of 11β-hydroxysteroid dehydrogenase type 1 in man: effects of prednisolone and chenodesoxycholic acid

The 11β-hydroxysteroid dehydrogenases (11β-HSDs) play a pivotal role in glucocorticoid (GC) action. 11β-HSD1 is a predominant reductase, activating GCs from inert metabolites, whereas 11β-HSD2 is a potent dehydrogenase inactivating GCs. Knowing the metabolic effects of GCs, a selective inhibition of 11β-HSD1 represents a potential target for therapy of impaired glucose tolerance, insulin insensitivity and central obesity. In vitro, 11β-HSD1 is selectively inhibited by chenodesoxycholic acid (CDCA) and upregulated under GC exposure. Therefore, we aimed to investigate the effects of CDCA and prednisolone on hepatic 11β-HSD1 activity in vivo by measuring 11-reduction of orally given cortisone (E) acetate to cortisol (F). CDCA or placebo was given to 5 male healthy volunteers within a randomised cross-over trial before and after oral administration of 12.5 mg E acetate at 8:00 h. For measurement of in vivo effects of GCs on 11β-HSD1 activity, hepatic reduction of 25 mg E acetate before and after treatment with prednisolone (30 mg for 6 days) was determined in 7 healthy males. Serum GC levels were determined using a fully automated liquid chromatographic system. CDCA had no effect on the activity of 11β-HSD1 in vivo. Prednisolone therapy leads to a marked rise in serum F concentrations and an elevated F/E serum ratio. This proves GC-induced activation of hepatic 11β-HSD1, which could not be extinguished by a parallel increase of IGF-1 under prednisolone. CDCA does not affect in vivo activity of 11β-HSD1 when given in therapeutic dosages. During GC treatment, increased hepatic activation of E to F may aggravate metabolic side effects of GCs such as seen in the metabolic syndrome.     

Comparison of total and salivary cortisol in a low-dose ACTH (Synacthen) test: influence of three-month oral contraceptives administration to healthy women

The objective of this study was to evaluate the influence of low-dose combined oral contraception (COC) on basal and stimulated (1 microg ACTH test) levels of serum and salivary cortisol (F), cortisone and on basal serum cortisol binding globulin (CBG), adrenocorticotropic hormone (ACTH), dehydroepiadrosterone (DHEA) and calculated free cortisol in healthy young women. Three-month administration of COC resulted in 1) significant increase of basal (454.0+/-125.0 to 860.9+/-179.7 nmol/l) and ACTH-stimulated serum cortisol in 30th min (652.3+/-60.5 to 1374.1+/-240.6 nmol/l); 2) no significant change of basal (15.4+/-7.3 to 18.9+/-8.5 nmol/l) and ACTH-stimulated salivary cortisol at the 30th min (32.4+/-8.8 to 32.9+/-9.0 nmol/l); 3) no significant change of basal serum cortisone (38,8+/-7.68 to 45.2+/-24.2 nmol/l) and ACTH-stimulated cortisone at the 30th (34.8+/-10.9 to 47.0+/-35.7 nmol/l); 4) significant increase of basal ACTH (17.2+/-9.0 to 38.2+/-29.4 ng/l), CBG (991.0+/-161.0 to 2332.0+/-428.0 nmol/l), and 5) no significant change of basal DHEA (24.6+/-15.7 to 22.6+/-11.7 micromol/l) and calculated basal value for free cortisol (22.8+/-14.9 to 19.2+/-6.9 nmol/l). In conclusions, higher basal and ACTH-stimulated serum cortisol were found after three-month administration of COC, while basal and stimulated salivary cortisol were not significantly affected. Therefore, salivary cortisol can be used for assessment of adrenal function in women regularly using COC.     

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC₅₀ value similar to that in in vitro microsomal assay (IC₅₀ = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.