Reduction Of Cortisone Research Articles

Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

Expression and activities of 11βHSD enzymes in the testes and reproductive tracts of sexually immature male pigs

In light of studies implicating glucocorticoids in the control of testicular steroidogenesis and/or spermatogenesis, the objective of this study was to characterise the expression and activities of the 11β-hydroxysteroid dehydrogenase (11βHSD) enzymes in the testis and reproductive tract of the pre-pubertal pig. Although 11βHSD1 and 11βHSD2 mRNA transcripts and proteins were co-expressed in all regions of the reproductive tract, cortisol–cortisone inter-conversion was detectable in the testis, caput epididymidis and bulbourethral glands only. In homogenates of these 3 tissues, the apparent K m for NADP +- and NAD +-dependent 11β-dehydrogenase activities ranged between 152–883 and 47–479 nmol l −1, respectively. Irrespective of the pyridine nucleotide co-substrate, estimates of V max were consistently two orders of magnitude higher in the testis. Moreover, while, in each tissue, levels of cortisol oxidation were comparable in the presence of either NADP + or NAD +, maximal rates of NAD(P) +-dependent cortisol oxidation were up to 33-fold greater than the V max for NADPH-dependent reduction of cortisone. We conclude that in the testis, caput epididymidis and bulbourethral gland of the immature pig, NADP +- and NAD +-dependent 11βHSD enzymes catalyse net inactivation of cortisol, suggesting a physiological role for these enzymes in limiting local actions of glucocorticoids within these male reproductive tissues prior to puberty.

Metyrapone prevents cortisone-induced preadipocyte differentiation by depleting luminal NADPH of the endoplasmic reticulum

Preadipocyte differentiation is greatly affected by prereceptorial glucocorticoid activation catalyzed by 11β-hydroxysteroid dehydrogenase type 1 in the lumen of the endoplasmic reticulum. The role of the local NADPH pool in this process was investigated using metyrapone as an NADPH-depleting agent. Metyrapone administered at low micromolar concentrations caused the prompt oxidation of the endogenous NADPH, inhibited the reduction of cortisone and enhanced the oxidation of cortisol in native rat liver microsomal vesicles. However, in permeabilized microsomes, it only slightly decreased both NADPH-dependent cortisone reduction and NADP +-dependent cortisol oxidation. Accordingly, metyrapone administration caused a switch in 11β-hydroxysteroid dehydrogenase activity from reductase to dehydrogenase in both 3T3-L1-derived and human stem cell-derived differentiated adipocytes. Metyrapone greatly attenuated the induction of 11β-hydroxysteroid dehydrogenase type 1 and the accumulation of lipid droplets during preadipocyte differentiation when 3T3-L1 cells were stimulated with cortisone, while it was much less effective in case of cortisol or dexamethasone. In conclusion, the positive feedback of glucocorticoid activation during preadipocyte differentiation is interrupted by metyrapone, which depletes NADPH in the endoplasmic reticulum. The results also indicate that the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum is important in the process of adipogenesis.

Leydig cells from neonatal pig testis abundantly express 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 and effectively inactivate cortisol to cortisone

11β-Hydroxysteroid dehydrogenase (11β-HSD) isozymes, designated types 1 and 2, catalyze the interconversion of physiologically active glucocorticoids and inactive 11-keto forms. The presence of types 1 and 2 was determined in neonatal pig testis and Leydig cells purified from testes by reverse transcription polymerase chain reaction, Western blotting, and immunohistochemical staining. Type 2 mRNA was expressed at a high level in neonatal pig testis. In particular, in the entire testis, a higher level of type 2 was expressed compared to type 1. Furthermore, these expression levels in the testis were compared with the expression levels of the respective isozymes in pig liver and kidney, which are known to have high levels. Next, the direction of glucocorticoid metabolism in intact Leydig cells was examined, and only oxidation from cortisol to cortisone was detected. Virtually no reduction of cortisone to cortisol was detected. Using a microsomal enzyme preparation from Leydig cells, type 2 exhibited potent oxidation activity, and the activity was higher than the oxidation activity catalyzed by the type 1 isozyme. In kinetic analysis, the K m and V max for type 1 were 1.36 μM and 0.91 nmol/(h mg), respectively, and 0.38 μM and 1.25 nmol/(h mg), respectively, for type 2. The results of the present study using neonatal pig testis suggest that not only 11β-HSD type 1 but also type 2, which is abundantly expressed, plays important roles in cortisol inactivation in pig Leydig cells, and furthermore, that excess cortisol will cause glucocorticoid-mediated suppression of testosterone production in even neonatal pig Leydig cells.

11β-Hydroxysteroid dehydrogenase enzymes in the testis and male reproductive tract of the boar (Sus scrofa domestica) indicate local roles for glucocorticoids in male reproductive physiology

11Beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11betaHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol-cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11betaHSD activities in the presence of NADPH/NADP(+) and NAD(+), and the penile urethra exhibited NAD(+)-dependent 11beta-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K(m) values of 237-443 and 154-226 nmol/l in the presence of NADP(+) and NAD(+) respectively. Maximal rates of NADP(+)-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V(max) for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD(+)-dependent cortisol metabolism. The relatively low K(m) estimates for NADP(+) -dependent cortisol oxidation suggest that either the affinity of 11betaHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP(+)-dependent 11betaHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.

Hexose-6-phosphate Dehydrogenase Modulates 11β-Hydroxysteroid Dehydrogenase Type 1-Dependent Metabolism of 7-keto- and 7β-hydroxy-neurosteroids

BackgroundThe role of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11β-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11β-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11β-HSD1 has not been assessed.MethodologyWe investigated the 11β-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7α-hydroxy- and 7β-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7β-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11β-HSD1.Principal FindingsWe demonstrated that 11β-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11β-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7β-hydroxy metabolites, indicating a role for H6PDH and 11β-HSD1 in the local generation of 7β-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7β-hydroxy-neurosteroids.ConclusionsOur results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11β-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11β-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

7α- and 7β-hydroxy-epiandrosterone as substrates and inhibitors for the human 11β-hydroxysteroid dehydrogenase type 1

The human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes both the NADP(H)-dependent oxido-reduction of cortisol and cortisone and the inter-conversion of 7α- and 7β-hydroxy-dehydroepiandrosterone (DHEA) through a 7-oxo-DHEA intermediate. As shown with human liver and intestine fractions, 7α-hydroxy-epiandrosterone (7α-hydroxy-EpiA) and 7β-hydroxy-EpiA were readily inter-converted with no evidence for a 7-oxo-EpiA intermediate. Whether this inter-conversion resulted from action of the 11β-HSD1 or from an unknown epimerase is unresolved. Furthermore, whether these steroids could inhibit the cortisol–cortisone oxido-reduction remains a question. The recombinant human 11β-HSD1 was used to test these questions. NADP + supplementation only provided the production of 7β-hydroxy-EpiA out of 7α-hydroxy-EpiA with a V max/ K M ratio at 0.1. With NADPH supplementation, both 7α-hydroxy-EpiA and 7β-hydroxy-EpiA were formed in low amounts from 7β-hydroxy-EpiA and 7α-hydroxy-EpiA, respectively. These inter-conversions occurred without a trace of the putative 7-oxo-EpiA intermediate. In contrast, the 7-oxo-EpiA substrate was efficiently reduced into 7α-hydroxy-EpiA and 7β-hydroxy-EpiA, with V max/ K M ratios of 23.6 and 5.8, respectively. Competitive and mixed type inhibitions of the 11β-HSD1-mediated cortisol oxidation were exerted by 7α-hydroxy-EpiA and 7β-hydroxy-EpiA, respectively. The 11β-HSD1-mediated cortisone reduction was inhibited in a competitive manner by 7-oxo-EpiA. These findings suggest that the active site of the human 11β-HSD1 may carry out directly the epimeric transformation of 7-hydroxylated EpiA substrates. The low amounts of these steroids in human do not support a physiological importance for modulation of the glucocorticoid status in tissues.

Gender‐specific Links Between Hepatic 11β Reduction of Cortisone and Adipokines

Reduction of cortisone to cortisol is mediated by 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1), a putative key enzyme in obesity-related complications. Experimental studies suggest that adipokines, notably leptin and tumor necrosis factor-alpha (TNF-alpha), are of importance for 11betaHSD1 activity. We hypothesized that the regulation of hepatic preceptor glucocorticoid metabolism is gender-specific and associated with circulating levels of leptin and TNF-alpha receptors and/or sex hormones. A total of 34 males and 38 women (14 premenopausal and 22 postmenopausal) underwent physical examination and fasting blood sampling. Insulin sensitivity was tested by euglycemic hyperinsulinemic clamps, and hepatic 11betaHSD1 enzyme activity was estimated by the conversion of orally-ingested cortisone to cortisol. Hepatic 11betaHSD1 activity was negatively associated with leptin and soluble TNF (sTNF) r1 and sTNFr2 in males. These correlations remained significant after adjustment for age and insulin sensitivity, and for sTNF-alpha receptors also after adjustment of BMI and waist circumference. In contrast, 11beta reduction of cortisone was positively associated to leptin in females after adjustment for BMI and waist circumference. Hepatic 11beta reduction shows different links to circulating adipocyte-derived hormones in males and females. This emphasizes the need for further studies on tissue-specific regulation of 11betaHSD1 in both genders.

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.

Role for Prostaglandins in the Regulation of Type 1 11β-Hydroxysteroid Dehydrogenase in Human Granulosa-Lutein Cells

11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes regulate glucocorticoid availability in target tissues. 11betaHSD1 is the predominant isoenzyme expressed and active in human granulosa-lutein (hGL) cells. This study investigated the effects of pharmacological inhibitors of prostaglandin (PG) synthesis on 11betaHSD1 activities and expression in hGL cells. The consequences for 11betaHSD1 of increasing exposure of hGL cells to PGs, either by treatment with exogenous PGs or by challenging cells with IL-1beta, were also assessed. Suppression of basal PG synthesis using four different inhibitors of PG H synthase enzymes [indomethacin, niflumic acid, meclofenamic acid (MA) and N-(2-cyclohexyloxy-4-nitorophenyl) methane sulfonamide (NS-398)] each resulted in significant decreases in both cortisol oxidation and cortisone reduction. Both activities of 11betaHSD1 were suppressed by up to 64+/-6% (P<0.05). Over 4 and 24 h, neither MA nor NS-398 affected the expression of 11betaHSD1 protein, suggesting enzyme regulation by PGs at the posttranslational level. When cells were cotreated for 4 h with PGHS inhibitors plus 30 nm PGD2, PGF2alpha, or PGE2, each PG overcame the suppression of cortisol oxidation by indomethacin or MA. Treatment of hGL cells with IL-1beta increased the concentrations of both PGE2 and PGF2alpha, accompanied by a 70+/-25% increase in net cortisol oxidation. All three responses to IL-1beta were abolished when cells were cotreated with MA. These findings suggest a role for PGs in the posttranslational regulation of 11betaHSD1 activities in hGL cells.

Abstracts for Oral Presentation

Abstracts for Oral Presentation

Inter-conversion of 7α- and 7β-hydroxy-dehydroepiandrosterone by the human 11β-hydroxysteroid dehydrogenase type 1

The dehydroepiandrosterone (DHEA) 7α-hydroxylation in humans takes place in the liver, skin, and brain. These organs are targets for the glucocorticoid hormones where 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates cortisone through its reduction into cortisol. The putative interference of 7α-hydroxy-DHEA with the 11β-HSD1-catalyzed reduction of cortisone into cortisol has been confirmed in preliminary works with human liver tissue preparations of the enzyme demonstrating the transformation of 7α-hydroxy-DHEA into 7-oxo-DHEA and 7β-hydroxy-DHEA. However, the large production of 7β-hydroxy-DHEA could not be explained satisfactorily. Therefore our objective was to study the role in the metabolism of oxygenated DHEA by recombinant human 11β-HSD1 expressed in yeast. The 7α- and 7β-hydroxy-DHEA were each oxidized into 7-oxo-DHEA with quite dissimilar K M (70 and 9.5 μM, respectively) but at equivalent V max. In contrast, the 11β-HSD1-mediated reduction of 7-oxo-DHEA led to the production of both 7α- and 7β-hydroxy-DHEA with equivalent K M (1.1 μM) but with a 7β-hydroxy-DHEA production characterized by a significantly greater V max. The 7α-hydroxy-DHEA produced by the cytochrome CYP7B1 in tissues may exert anti-glucocorticoid effects through interference with the 11β-HSD1-mediated cortisone reduction.

Obesity and Cortisol Status

The fact, that obesity is a prominent feature of hypercortisolism (Cushing's syndrome) has stimulated investigation on the possible existence of the reverse relationship, namely that hypercortisolism is a feature of obesity. We have reviewed half a century of literature on this question, and have found out the following: (1) Hypercortisolism can exist in two forms: systemic hypercortisolism, in which there is an overall bodily excess of cortisol, and tissue, or intracellular, hypercortisolism, in which there is increased intracellular concentration of cortisol without an overall bodily excess. (2) There are two parameters of systemic hypercortisolism: CPR and plasma cortisol concentration. Proper evaluation of the first parameter requires correction for the active metabolic mass, which is best performed by expressing CPR per gram of urinary creatinine. The second parameter can be confounded by the marked moment-to-moment fluctuations in plasma cortisol concentrations due to cortisol's episodic secretion. Proper evaluation requires measuring the 24-hour mean concentration. Of these two parameters of systemic cortisol status, the plasma concentration is the more critical and accurate. (3) Corrected CPR is normal in obese individuals, and 24-hour mean plasma cortisol concentrations are slightly but definitely subnormal. This combination of findings indicates diminished stimulability of the hypothalamic-pituitary-adrenal (HPA) axis, which normally regulates bodily cortisol status. This deduction is supported by empirical studies on HPA reactivity. (4) Tissue hypercortisolism, due to increased intracellular activity of 11beta-HSD-1, which catalyzes reduction of cortisone to cortisol, has been reported in obese mice and humans. The findings of various studies are not consistent, and whether the enzymatic overactivity is a cause or a result of obesity is still unclear.

Cooperativity between 11β-Hydroxysteroid Dehydrogenase Type 1 and Hexose-6-phosphate Dehydrogenase in the Lumen of the Endoplasmic Reticulum

The functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase was investigated in rat liver microsomal vesicles. The activity of both enzymes was latent in intact vesicles, indicating the intraluminal localization of their active sites. Glucose-6-phosphate, a substrate for hexose-6-phosphate dehydrogenase, stimulated the cortisone reductase activity of 11beta-hydroxysteroid dehydrogenase type 1. Inhibition of glucose-6-phosphate uptake by S3483, a specific inhibitor of the microsomal glucose-6-phosphate transporter, decreased this effect. Similarly, cortisone increased the intravesicular accumulation of radioactivity upon the addition of radiolabeled glucose-6-phosphate, indicating the stimulation of hexose-6-phosphate dehydrogenase activity. A correlation was shown between glucose-6-phosphate-dependent cortisone reduction and cortisone-dependent glucose-6-phosphate oxidation. The results demonstrate a close cooperation of the enzymes based on co-localization and the mutual generation of cofactors for each other.

Mutations in the genes encoding 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase interact to cause cortisone reductase deficiency.

In cortisone reductase deficiency (CRD), activation of cortisone to cortisol does not occur, resulting in adrenocorticotropin-mediated androgen excess and a phenotype resembling polycystic ovary syndrome (PCOS; refs. 1,2). This suggests a defect in the gene HSD11B1 encoding 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), a primary regulator of tissue-specific glucocorticoid bioavailability. We identified intronic mutations in HSD11B1 that resulted in reduced gene transcription in three individuals with CRD. In vivo, 11beta-HSD1 catalyzes the reduction of cortisone to cortisol whereas purified enzyme acts as a dehydrogenase converting cortisol to cortisone. Oxo-reductase activity can be regained using a NADPH-regeneration system and the cytosolic enzyme glucose-6-phosphate dehydrogenase. But the catalytic domain of 11beta-HSD1 faces into the lumen of the endoplasmic reticulum (ER; ref. 6). We hypothesized that endolumenal hexose-6-phosphate dehydrogenase (H6PDH) regenerates NADPH in the ER, thereby influencing directionality of 11beta-HSD1 activity. Mutations in exon 5 of H6PD in individuals with CRD attenuated or abolished H6PDH activity. These individuals have mutations in both HSD11B1 and H6PD in a triallelic digenic model of inheritance, resulting in low 11beta-HSD1 expression and ER NADPH generation with loss of 11beta-HSD1 oxo-reductase activity. CRD defines a new ER-specific redox potential and establishes H6PDH as a potential factor in the pathogenesis of PCOS.

Fetal lung maturation in estrogen-deprived baboons.

We have previously shown that estrogen plays a central integrative role in regulating key aspects of fetal-placental development and that inhibition of estrogen production during the second half of baboon pregnancy suppressed fetal adrenal function. Because maturation of the fetal lung is dependent on cortisol of fetal adrenal origin, the current study determined whether lung development and expression of surfactant proteins (SPs) A and B were altered at term in estrogen-deprived baboons. Fetal lungs were obtained on d 100, 165, and 175 of gestation (term = d 184) from untreated baboons and on d 165 from animals treated daily during the second half of pregnancy either with the aromatase inhibitor CGS 20267 alone or with CGS 20267 and estradiol benzoate. Umbilical venous estradiol levels were suppressed by more than 95% by CGS 20267 and elevated by CGS 20267 and estrogen. Although umbilical serum cortisol levels were also suppressed by 35% by CGS 20267, cortisol levels in the fetal lung of estrogen-suppressed baboons were similar to values in untreated animals. Immunocytochemistry demonstrated that CGS 20267 treatment did not alter fetal lung expression of the 11 beta-hydroxysteroid dehydrogenase enzyme-1 enzyme catalyzing reduction of cortisone to cortisol. However, immunocytochemical expression of the 11 beta-hydroxysteroid dehydrogenase enzyme-2 catalyzing oxidation of cortisol to cortisone appeared lower in lungs of estrogen-deprived fetuses and restored to normal by CGS 20267 and estrogen. SP-A levels in fetal lungs of untreated baboons were increased 16- to 20-fold between d 100 and d 165-175 of gestation in untreated baboons and baboons treated with CGS 20267 or CGS 20267 and estrogen. Similarly, SP-B levels in fetal lungs of untreated baboons were increased 10-fold between d 100 and d 165-175 of gestation in untreated baboons and baboons treated with CGS 20267 or CGS 20267 and estrogen. Moreover, in estrogen-suppressed baboons, as in untreated animals, the fetal lung continued to grow and exhibited normal alveolarization on histology. We conclude that development of the primate fetal lung can occur in utero in baboons in which fetal serum cortisol levels have been suppressed by the relative absence of estrogen perhaps because of the ability of the lung to coordinate local production of cortisol.

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity in follicular fluid from gonadotrophin-stimulated assisted conception cycles

In the ovary, cortisol-cortisone interconversion is catalysed by isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to establish whether human follicular fluid (hFF), obtained after controlled ovarian hyperstimulation, contains paracrine modulators of 11betaHSD activity. Of 274 hFF samples tested for effects in rat kidney homogenates, 206 hFF samples significantly inhibited NADP(+)-dependent oxidation of cortisol within 1 h (by 11-67% of control 11betaHSD activity), whereas 42 hFF samples significantly stimulated 11betaHSD activity (16-210% increase relative to control). Although charcoal-stripping of hFF prevented the inhibition and potentiated the stimulation of NADP(+)-dependent cortisol oxidation in a renal homogenate, effects of individual hFF samples on NADP(+)-dependent cortisol oxidation were independent of intrafollicular progesterone concentrations. Hydrophilic fractions of hFF samples, isolated by C18 column chromatography, stimulated both the NADP(+)-dependent oxidation of cortisol (by 55+/-5%, n=98) and the NADPH-dependent reduction of cortisone (by 86+/-22%, n= 5). In contrast, the hydrophobic fractions of hFF (eluted at 65-85% methanol) inhibited both NADP(+)-dependent 11beta-dehydrogenase and NADPH-dependent 11-ketosteroid reductase activities (by 63+/-2% and 74+/-4%, respectively). None of the C18 column fractions of 50 hFF samples had any significant effect on NAD(+)-dependent 11beta-dehydrogenase activities. The hydrophobic inhibitors of NADP(H)-dependent cortisol-cortisone metabolism did not co-elute with several candidate compounds (prostaglandins E(2) and F(2alpha), cortisol, cortisone, oestradiol, testosterone, progesterone, pregnenolone or cholesterol). Hence, hFF aspirated from women undergoing controlled ovarian hyperstimulation for assisted conception contains both hydrophilic stimuli and hydrophobic inhibitors of glucocorticoid metabolism which appear to be selective for the NADP(H)-dependent, type 1 isoform of 11betaHSD.

In the search for specific inhibitors of human 11beta-hydroxysteroid-dehydrogenases (11beta-HSDs): chenodeoxycholic acid selectively inhibits 11beta-HSD-I.

Selective inhibitors of 11beta-hydroxysteroid-dehydrogenase type I may be of therapeutical interest for two reasons: i) 9alpha-Fluorinated 11-dehydrosteroids like 11-dehydro-dexamethasone (DH-D) are rapidly activated by human kidney 11beta-hydroxysteroid-dehydrogenase type II (11beta-HSD-II) to dexamethasone (D). If the same reaction by hepatic 11beta-HSD-I could be selectively inhibited, DH-D could be used for selective renal immunosuppressive therapy. ii) Reduction of cortisone to cortisol in the liver may increase insulin resistance in type 2 diabetes mellitus, and inhibition of the enzyme may lead to a decrease in gluconeogenesis. Therefore, we characterized the metabolism of DH-D by human hepatic 11beta-HSD-I and tried to find a selective inhibitor of this isoenzyme. For kinetic analysis of 11beta-HSD-I, we used microsomes prepared from unaffected parts of liver segments, resected because of hepatocarcinoma or metastatic disease. For inhibition experiments, we also tested 11beta-HSD-II activity with human kidney cortex microsomes. The inhibitory potency of several compounds was evaluated for oxidation and reduction in concentrations from 10(-9) to 10(-5)mol/l. Whereas D was not oxidized by human liver microsomes at all, cortisol was oxidized to cortisone with a maximum velocity (V(max)) of 95pmol/mg per min. The reduction of DH-D to D (V(max)=742pmol/mg per min, Michaelis--Menten constant (K(m))=1.6 micromol/l) was faster than that of cortisone to cortisol (V(max)=187pmol/mg per min). All reactions tested in liver microsomes showed the characteristics of 11beta-HSD-I: K(m) values in the micromolar range, preferred cosubstrate NADP(H), no product inhibition. Of the substances tested for inhibition of 11beta-HSD-I and -II, chenodeoxycholic acid was the only one that selectively inhibited 11beta-HSD-I (IC(50) for reduction: 2.8x10(-6)mol/l, IC(50) for oxidation: 4.4x10(-6)mol/l), whereas ketoconazole preferentially inhibited oxidation and reduction reactions catalyzed by 11beta-HSD-II. Metyrapone, which is reduced to metyrapol by hepatic 11beta-HSD-I, inhibited steroid reductase activity of 11beta-HSD-I and -II and oxidative activity of 11beta-HSD-II. These findings can be explained by substrate competition for reductase reactions and by product inhibition of the oxidation, which is a well-known characteristic of 11beta-HSD-II. Our in vitro results may offer a new concept for renal glucocorticoid targeting. Oral administration of small amounts of DH-D (low substrate affinity for 11beta-HSD-I) in combination with chenodeoxycholic acid (selective inhibition of 11beta-HSD-I) may prevent hepatic first pass reduction of DH-D, thus allowing selective activation of DH-D to D by the high affinity 11beta-HSD-II in the kidney. Moreover, selective inhibitors of the hepatic 11beta-HSD-I, like chenodeoxycholic acid, may become useful in the therapy of patients with hepatic insulin resistance including diabetes mellitus type II, because cortisol enhances gluconeogenesis.

Evidence for isoforms of 11 beta-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig.

In the human and in rodents like the rat and mouse, the liver enzyme 11 beta-hydroxysteroid dehydrogenase type I (11 beta-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11 beta-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular, mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11 beta-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10(-6) mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10(-3) mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with K(m) values of 0.64 mumol/l and 9.8 mumol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate K(m) was 7.2 mumol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar K(m) and Vmax values for NAD+ to NADP+ as cosubstrates (K(m) of 4.3 mumol/l and 5.0 mumol/l respectively). The NAD+ preference for the oxidation of 10(-6) mol/l cortisol described above may be due to a second, NAD(+)-preferring 11 beta-HSD with a K(m) of 1.4 mumol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (K(m): 1.2 mumol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11 beta-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD(+)-preferring. This kidney enzyme (analogous to 11 beta-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.

Comparison of 11β-hydroxysteroid dehydrogenase in spontaneously hypertensive and Wistar-Kyoto rats

11β-Hydroxysteroid dehydrogenase (11β-HSD) modulates glucocorticoid interactions with mineralocorticoid and glucocorticoid receptors in vivo, by converting 11β-hydroxyglucocorticoids to their inactive 11-ketone derivatives. Defective 11β-oxidation of glucocorticoids has been associated with hypertension. The objective of this study was to investigate whether 11β-HSD contributes to the occurrence of hypertension in spontaneously hypertensive rats (SHRs). The liver and kidney microsomal oxidations of corticosterone (the physiological glucocorticoid in rats) in organs from juvenile (3 weeks old) and adult (3 months old) SHR and Wistar-Kyoto (WKY) rats, with NAD and NADP, show no differences between rat strains. For cortisol, with NADP, adult SHRs show (1.3–3 times; P < 0.05) lower kidney microsomal oxidation rates. The liver microsomal reduction of cortisone shows remarkable interstrain differences: with NADH, reduction is conducted only by adult WKY rats, whereas with NADPH, juvenile animals show similar reduction rates, but at adulthood, only WKYs reduce cortisone. Using Western blot analysis with antibodies against 11β-HSD1, positive signals are obtained only for liver microsomes, appearing somewhat lower in SHRs for juvenile but not adult animals. Urinary corticosterone/11-dehydrocorticosterone ratios (measured in adult animals) are not different between rat strains, but are elevated after administration of corticosterone in both strains (although significant only in SHRs). The data provide no indications for exaggerated stimulation of renal corticosteroid receptors, due to modified 11β-HSD, in SHRs. However, the experiments suggest the existence of multiple 11β-HSDs, in addition to 11β-HSD1 and 11β-HSD2, some of which may be modified in SHR, but the nature and physiological role of these 11β-HSDs is unclear.