beta-HSD Activity Research Articles

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.

Osteoblastic 11beta-hydroxysteroid dehydrogenase type 1 activity increases with age and glucocorticoid exposure.

The risk of glucocorticoid-induced osteoporosis increases substantially with age but there is considerable individual variation. In recent studies we have shown that the effects of glucocorticoids on bone are dependent on autocrine actions of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1); expression of 11beta-HSD1 in osteoblasts (OBs) facilitates local synthesis of active glucocorticoids with consequent effects on osteoblastic proliferation and differentiation. Using primary cultures of human OBs, we have now characterized the age-specific variation in osteoblastic 11beta-HSD1 and defined enzyme kinetics and regulation using natural and therapeutic glucocorticoids. 11beta-HSD1 reductase activity (cortisone to cortisol conversion) was recognized in all OB cultures and correlated positively with age (r = 0.58 with all cultures, p < 0.01, and n = 18; r = 0.87 with calcaneal-derived cultures, p < 0.001, and n = 14). Glucocorticoid treatment caused a time- and dose-dependent increase in 11beta-HSD1 activity over control (e.g., dexamethasone [DEX; 1 microM], 2.6-fold +/- 0.5 (mean +/- SE), p < 0.001, and n = 16; cortisol (100 nM), 1.7-fold +/- 0.1, p < 0.05, and n = 14). Similar increases in 11beta-HSD1 mRNA expression were indicated using real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses (3.5-fold with DEX, p < 0.01; 2.5-fold with cortisol, p < 0.05). The capacity of 11beta-HSD1 to metabolize the synthetic glucocorticoids prednisone and prednisolone was investigated in human OBs (hOBs) and fetal kidney-293 cells stably transfected with human 11beta-HSD1 cDNA. Transfected cells and hOBs were able to interconvert prednisone and prednisolone with reaction kinetics indistinguishable from those for cortisone and cortisol. To assess the in vivo availability of substrates for osteoblastic 11beta-HSD1, plasma cortisone and prednisone levels were measured in normal males before and after oral prednisolone (5 mg). The 9:00 a.m. serum cortisone levels were 110 +/- 5 nmol/liter and prednisone levels peaked at 78 +/- 23 nmol/liter 120 minutes after administration of prednisolone. Thus, therapeutic use of steroids increases substrate availability for 11beta-HSD1 in bone. These studies indicate that activation of glucocorticoids at an autocrine level within bone is likely to play an important role in the age-related decrease in bone formation and increased risk of glucocorticoid-induced osteoporosis.

Glucocorticoids and 11 beta-hydroxysteroid dehydrogenase type 2 gene expression in the aging kidney.

Aging is associated with increased concentrations of circulating glucocorticoids, a situation expected to induce a glucocorticoid-mediated mineralocorticoid effect, resulting in sodium retention and hypertension unless counteracting mechanisms are operative. Conversion of glucocorticoids to inert 11 beta-keto compounds by the enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) is one of these mechanisms. We hypothesized therefore that 11 beta-HSD2 gene expression and/or activity increase with age in male WAG/Rij rats, a strain without increased blood pressure with age or senescence-related obesity or kidney disease. Corticosterone (B) concentrations in plasma and urinary excretion of corticosterone and dehydrocorticosterone (A) tetrahydro metabolites, THB + 5 alpha-THB + THA, were assessed by gas chromatography-mass spectrometry (GC-MS) in 10-month-old-rats (n = 6) and in 30-month-old rats (n = 6). Renal 11 beta-HSD2 messenger ribonucleic acid (mRNA) abundance was measured by real-time quantitative TaqMan polymerase chain reaction and microarray assays. Thirty-month-old rats had significantly higher corticosterone concentrations in plasma and increased urinary excretion of corticosterone and dehydrocorticosterone tetrahydro metabolites. Conversion of B to A in kidney microsomes from 30-month-old rats was moderately but not significantly increased compared with 10-month-old rats. The urinary ratios of (THB + 5 alpha-THB)/THA and free B/A and renal 11 beta-HSD2 mRNA abundance were equal in 10- and 30-month-old rats. There is no evidence for an enhanced gene expression or activity of renal 11 beta-HSD2 in these aging rats, suggesting either that endogenous 11 beta-HSD2 is able to cope with the increased corticosterone concentrations characteristic of the aging process or that alternative mechanisms contribute to the maintenance of a normal sodium excretion in these animals.

Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency.

To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 +/- 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.

Inhibition of 3beta-hydroxysteroid dehydrogenase-isomerase in mouse adrenal cells: a direct effect of testosterone.

Gonadal steroids modulate adrenal gland size and function in a variety of species, and our previous studies demonstrate that circulating androgens suppress 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) activity in the adrenal cortex of male mice. The present study tests the hypothesis that androgens have a direct, receptor-mediated inhibitory effect on adrenal 3betaHSD. Treatment of cultured adrenal cells from C57BL/6J and C3H/HeJ mice with 0.02-2.0 microM testosterone for 7 days significantly reduces 3betaHSD activity in cells from both strains. However, treatment for 3 days reduces 3betaHSD activity in the adrenal cells from C3H/HeJ, but not C57BL/6J mice. The decreases in 3betaHSD activity in response to testosterone treatment is reflected in decreases in the amount of 3betaHSD immunoreactive protein, such that extended treatment decreases 3betaHSD immunoreactive protein in adrenal cells from both strains, but short-term treatment only decreases 3betaHSD immunoreactive protein in adrenal cells from C3H/HeJ mice. Thus, there appears to be a temporal difference between strains in the effect of the testosterone on 3betaHSD activity and immunoreactive protein. Treatment of the adrenal cells with androgen agonists and an antagonist indicate that the effect of testosterone is androgen receptor mediated. The effect of testosterone appears to be specific for 3betaHSD, since none of the treatments alter P450(scc) in cells from either strain. Testosterone treatment also causes a decrease in the amount of 3betaHSD mRNA. However, in contrast to the effect on activity and immunoreactive protein, there is no strain-related temporal difference because testosterone decreases 3betaHSD mRNA within 24h in adrenal cells from both strains. These results indicate that testosterone can act directly on the adrenal gland to decrease 3betaHSD activity, immunoreactive protein, and mRNA content in mouse adrenal glands, and thus contribute to the sex difference in adrenal function observed in many species.

Human adrenal cortex and aldosterone secreting adenomas express both 11β-hydroxysteroid dehydrogenase type 1 and type 2 genes

11beta-Hydroxysteroid dehydrogenase type 1 and type 2 (11betaHSD1 and 11betaHSD2) isozymes catalize the conversion of inactive glucocorticoids (e.g. cortisone) to their active forms (e.g. cortisol) and vice versa, respectively. Reverse transcription-polymerase chain reaction allowed the detection of 11betaHSD1 and 11betaHSD2 mRNAs in the human adrenal cortex, liver, kidneys, as well as in six aldosterone-secreting adenomas. 11betaHSD1 and 11betaHSD2 activity, as evaluated by the capacity of the microsomal fraction to convert [3H]cortisone to [3H]cortisol and vice versa, was detected in both human adrenal cortex and aldosteronomas, although it was less elevated than in liver and kidneys. Aldosteronomas possessed more intense 11betaHSD1 activity and less intense 11betaHSD2 activity than the normal adrenal cortex. The hypothesis is advanced that the elevated local concentration of steroid-hormone intermediates occurring in aldosteronomas up-regulates 11betaHSD1 and down-regulates 11betaHSD2, thereby contributing to their enhanced steroidogenic function.

Reduced 11beta-hydroxysteroid dehydrogenase activity in experimental nephrotic syndrome.

The disease state of the nephrotic syndrome is characterized by abnormal renal sodium retention that cannot be completely explained by a secondary hyperaldosteronism for the following reasons. Firstly, in rats an enhanced sodium retention is observed before proteinuria with intravascular volume depletion occurs. Secondly, in patients with the nephrotic syndrome, volume expansion with hypertension has been reported despite suppression of the renin-aldosterone system. Therefore, another mechanism for sodium retention must be postulated for this disease state. We hypothesize that this mechanism is a reduced 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) activity, a phenomenon known to cause enhanced access of cortisol or corticosterone to the mineralocorticoid receptor. We assessed the 11beta-HSD activity by measuring the urinary ratio of tetrahydrocorticosterone (THB) plus 5alpha-tetrahydrocorticosterone (5alpha-THB) to 11-dehydro-tetrahydrocorticosterone (THA) by gas chromatography-mass spectrometry in rats with puromycin aminonucleoside (PAN)-induced proteinuria and with adriamycin nephrosis. Furthermore, the plasma ratios of corticosterone to 11-dehydrocorticosterone were measured. The urinary ratio of (THB+5alpha-THB)/THA increased in all animals following injection of PAN or adriamycin, indicating a reduced activity of 11beta-HSD. The reduced activity of 11beta-HSD was confirmed by an increased plasma ratio of corticosterone to 11-dehydrocorticosterone. The changes in the glucocorticoid metabolite ratios were already present before significant proteinuria appeared. PAN- or adriamycin-treated rats develop proteinuria with a reduced activity of 11beta-HSD, a mechanism contributing to the abnormal sodium retention in nephrotic syndrome.

Increased cortisol metabolites and reduced activity of 11β-hydroxysteroid dehydrogenase in patients on hemodialysis

Patients with renal failure have symptoms assumed to be attributable to the accumulation of toxic endo- or xenobiotics. Most of these molecules, especially those with a molecular weight>300 D, have not been identified. In addition to excretion, the kidney is involved in some defined metabolic processes. In the cortical collecting duct, the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) interconverts cortisol (F) and cortisone (E), and the metabolites of these glucocorticoids, tetrahydrocortisol (THF), 5alpha-tetrahydrocortisol (5alpha-THF) and tetrahydrocortisone (THE), are excreted in urine. We hypothesized that first, these metabolites accumulate and second, their concentration pattern changes in patients on hemodialysis. THF, 5alpha-THF, THE, F and E were measured in plasma of 63 patients on dialysis and in 34 healthy controls by gas-chromatography-mass spectrometry (GC/MS). In 11 patients, the metabolite clearance was determined during high flux hemodialysis by using a population pharmacokinetic approach. Mean plasma concentrations of THF, 5alpha-THF and THE were more than five times higher and those of E lower in patients than in controls. The ratios of (THF + 5alpha-THF)/THE and F/E were increased in patients, indicating a reduced activity of 11beta-HSD2. Intradialytic clearances were between 120 and 300 mL/min and not sufficient to normalize the steroid concentrations. Patients on hemodialysis exhibit pronounced increases in THF, 5alpha-THF and THE concentrations in plasma with insufficient removal during dialysis. Due to a reduced 11beta-HSD2 activity, an abnormal pattern of the concentrations of these cortisol and cortisone metabolites is observed. Since many signs and symptoms in uremic patients resemble those observed in subjects with glucocorticoid excess, the clinical relevance of the high concentrations of these glucocorticoid metabolites deserves further investigation.

Reduced 11β-Hydroxysteroid Dehydrogenase Type 2 Activity Is Associated with Decreased Birth Weight Centile in Pregnancies Complicated by Asthma

Pregnancies complicated by asthma are associated with an increased risk of low birth weight. Currently, the mechanisms causing this outcome are unknown. To investigate whether impaired placental function may be a determinant, we measured placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity, protein and mRNA, placental CRH mRNA, fetal cortisol, and fetal estriol concentrations at delivery. Asthmatic subjects were classified according to inhaled glucocorticoid intake during pregnancy and compared with a control nonasthmatic group. There was a 25% reduction in neonatal birth weight centile in asthmatic women who did not use inhaled glucocorticoid treatment. This was accompanied by significantly reduced placental 11beta-HSD2 activity, significantly increased fetal cortisol, and a trend toward increased placental CRH mRNA and reduced fetal estriol concentrations. The use of inhaled glucocorticoids for treatment was associated with birth weight centile, 11beta-HSD2 activity, CRH mRNA, fetal cortisol, and estriol concentrations similar to control levels. There was a significant inverse correlation between fetal cortisol and fetal estriol concentrations across all groups. These studies demonstrate that inhaled glucocorticoid intake for the treatment of asthma is associated with improved placental function and fetal outcome, suggesting that inflammatory factors associated with asthma may be detrimental to fetal growth and development in these pregnancies.

Association studies between the HSD11B2 gene (encoding human 11beta-hydroxysteroid dehydrogenase type 2), type 1 diabetes mellitus and diabetic nephropathy.

Mutations in the HSD11B2 gene (encoding human 11beta-hydroxysteroid dehydrogenase type 2) explain the syndrome of apparent mineralocorticoid excess where cortisol acts as a mineralocorticoid. A microsatellite marker within the HSD11B2 gene associates with salt sensitivity and hypertension--phenotypes characterising diabetic nephropathy. Here, we evaluate the HSD11B2 gene as a susceptibility locus for diabetic nephropathy. 150 patients with type 1 diabetes and nephropathy (DN), 145 patients with type 1 diabetes with a long duration of non-nephropathy (LDNN) and 151 normal controls were studied. We determined allele frequencies for the (CA)n repeat marker within intron I of the HSD11B2 gene. Duration of type 1 diabetes, diabetic status and renal function were recorded. 11 alleles (138-158) for the marker were observed. Allele 152 was significantly increased in controls compared with LDNN (70.5% vs 57.6%, P(c)<0.05 where P(c) is the P value corrected for multiple comparisons) but no difference was observed between DN and LDNN subjects. Allele 154 was significantly increased in the LDNN compared with the DN subjects (15.9% vs 7.0%, P(c)<0.01) but no difference was observed between DN and controls. A greater proportion of subjects carried at least 1 allele <152 in DN compared with control subjects (47.3% vs 28.5%, P(c)<0.01), but no difference was observed in LDNN compared with control and DN subjects. Weak associations are reported between the HSD11B2 gene, type 1 diabetes mellitus and nephropathy. The increased frequency of HSD11B2 short alleles in the diabetic groups may reflect reduced renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity and may, in part, explain the enhanced salt sensitivity observed in patients with type 1 diabetes.

Expression of steroid-converting enzymes in osteoblasts derived from rat vertebrae.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 activity in ovine placenta by fetal cortisol.

The effect of fetal cortisol on the activity of the type 2 isoform of the enzyme, 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2), was examined in ovine placenta and fetal kidney by measuring tissue 11 beta-HSD2 activity during late gestation when endogenous fetal cortisol levels rise and after exogenous cortisol administration to immature fetuses before the prepartum cortisol surge. Placental 11 beta-HSD2 activity decreased between 128-132 days and term (approximately 145 days of gestation) in association with the normal prepartum increase in fetal plasma cortisol. Raising fetal cortisol levels to prepartum values in the immature fetus at 128--132 days of gestation reduced placental 11 beta-HSD2 activity to term values. In contrast, 11 beta-HSD2 activity in the fetal renal cortex was unaffected by gestational age or cortisol infusion. When all the data were combined, there was an inverse correlation between the log fetal plasma cortisol level at delivery and placental 11 beta-HSD2 activity, expressed both on a weight-specific basis and per mg placental protein. Fetal cortisol therefore appears to be a physiological regulator of placental, but not renal, 11 beta-HSD2 activity in fetal sheep during late gestation. These findings have important implications, not only for glucocorticoid exposure in utero, but also for the local actions of cortisol within the placental tissues that are involved in initiating parturition in the sheep.

A switch in dehydrogenase to reductase activity of 11 beta-hydroxysteroid dehydrogenase type 1 upon differentiation of human omental adipose stromal cells.

As exemplified in patients with Cushing's syndrome, glucocorticoids play an important role in regulating adipose tissue distribution and function, but circulating cortisol concentrations are normal in most patients with obesity. However, human omental adipose stromal cells (ASCs) can generate glucocorticoid locally through the expression of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 1 (11 beta-HSD1), which, in intact cells, has been considered to be an oxoreductase, converting inactive cortisone (E) to cortisol (F). Locally produced F can induce ASC differentiation, but the relationship between 11 beta-HSD1 expression and adipocyte differentiation is unknown. Primary cultures of paired omental (om) and sc ASC and adipocytes were prepared from 17 patients undergoing elective abdominal surgery and cultured for up to 14 d. Expression and activity of 11 beta-HSD isozymes were analyzed together with early (lipoprotein lipase) and terminal (glycerol 3 phosphate dehydrogenase) markers of adipocyte differentiation. On d 1 of culture, 11 beta-HSD1 activity in intact om ASCs exceeded oxoreductase activity in every patient (78.9 +/- 24.9 vs. 15.8 +/- 3.7 [mean +/- SE] pmol/mg per hour, P < 0.001), and in sc ASCs, relative activities were similar (40.6 +/- 12.2 vs. 36.9 +/- 8.8). Conversely, in freshly isolated om adipocytes, reductase activity exceeded dehydrogenase activity (23.6 +/- 1.5 vs. 6.2 +/- 0.8 pmol/mg per hour, P < 0.01). Following 14 d of culture in serum-free conditions with addition of 10 nM insulin (Ctr) or insulin with 100 nM F (+F), lipoprotein lipase/18S RNA levels increased in both the Ctr- and +F-treated ASCs, but glycerol 3 phosphate dehydrogenase increased only in the +F cultures. In both cases, however, 11 beta-HSD1 oxoreductase activity exceeded dehydrogenase activity (Ctr: 53.3 +/- 9.0 vs. 32.4 +/- 10.5, P < 0.05; +F: 65.6 +/- 15.6 vs. 37.1 +/- 11.5 pmol/mg per hour, P < 0.05), despite no significant changes in 11 beta-HSD1 mRNA levels. In sc ASCs, dehydrogenase activity was similar to reductase activity in both Ctr- and +F-treated cells. Type 2 11 beta-HSD expression was undetectable in each case. These data show that in intact, undifferentiated om ASCs, 11 beta-HSD1 acts primarily as a dehydrogenase, but in mature adipocytes oxoreductase activity predominates. Because glucocorticoids inhibit cell proliferation, we postulate that 11 beta-HSD1 activity in uncommitted ASCs may facilitate proliferation rather than differentiation. Once early differentiation is initiated, a "switch" to 11 beta-HSD1 oxoreductase activity generates F, thus promoting adipogenesis. Site-specific regulation of the set-point of 11 beta-HSD1 activity may be an important mechanism underpinning visceral obesity.

11 beta-Hydroxysteroid dehydrogenase antisense affects vascular contractile response and glucocorticoid metabolism.

Glucocorticoids (GC's) are metabolized in vascular tissue by two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). 11 beta-HSD2 is unidirectional and metabolizes GC's to their respective inactive 11-dehydro derivatives. 11 beta-HSD1 is bi-directional, also possessing reductase activity and thus the ability to regenerate active GC from the 11-dehydro derivatives. In vascular tissue, GC's amplify the pressor responses to catecholamines and angiotensin II and may down-regulate certain depressor systems such as nitric oxide and prostaglandins. We hypothesize that both 11 beta-HSD2 and 11 beta-HSD1 regulate GC levels in vascular tissue and are part of additional mechanisms that control vascular tone. We examined the effects of specific antisense oligomers to 11 beta-HSD2 and 11 beta-HSD1 on GC metabolism and contractile response to phenylephrine (PE) in rat aortic rings. In aortic rings incubated (24 h) with corticosterone (B) (10 nmol/l) and 11 beta-HSD2 antisense (3 micromol/l), the contractile response to graded concentrations of PE (PE: 10 nmol/l - 1 micromol/l) were significantly (P < 0.05) increased compared to rings incubated with B and 11 beta-HSD2 nonsense. 11 beta-HSD1 antisense oligomers also enhanced the ability of B to amplify the contractile response to PE. In addition, 11 beta-HSD2 and 11 beta-HSD1 antisense also decreased the metabolism of B to 11-dehydro-B. 11-Dehydro-B (100 nmol/l) also amplified the contractile response to PE in aortic rings (P < 0.01), most likely due to the generation of active corticosterone by 11 beta-HSD1-reductase; this effect was significantly attenuated by 11 beta-HSD1 antisense. 11 beta-HSD1 antisense also caused a marked decrease in the metabolism of 11-dehydro-B back to B by 11 beta-HSD1-reductase. These findings underscore the importance of 11 beta-HSD2 and 11 beta-HSD1 in regulating local concentrations of GC's in vascular tissue. They also indicate that decreased 11 beta-HSD2 activity may be a possible mechanism in hypertension and that 11 beta-HSD1-reductase may be a possible target for anti-hypertensive therapy.

Expression of 3 beta-hydroxysteroid dehydrogenase in early bovine embryo development.

We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5-->4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro produced bovine embryos. 3 beta-HSD activity was detected in in vitro matured oocytes (74.4 +/- 1.4%), 1-cell (72.9 +/- 6.1%), 2-cell (61.8 +/- 7.4%), 8-cell (50 +/- 5%), morulae (50.8 +/- 2.6%), blastocysts (94.4 +/- 3%), and hatched blastocysts (100 +/- 0%) meanwhile the 4-cell stage showed a significant reduction (16.7 +/- 4.7%). When total embryonic RNA of different stages was subjected to RT-PCR assays, the mRNA of 3 beta-HSD was found to be present in all developmental stages of in vitro produced bovine embryos, from the oocyte to the blastocyst, with a marked decrease at the 4-cell stage. To determine whether the temporal pattern of enzyme activity was dependent on the maternal to zygotic transition, embryos were incubated in the presence of a transcription inhibitor, alpha-amanitin. The reappearance of the enzyme activity after the 4-cell stage was blocked in alpha-amanitin treated embryos, indicating the requirement of embryonic transcription. On the other hand, the embryonic steroid metabolism was tested by incubating blastocyst with tritiated pregnenolone. Analysis of the metabolites by TLC indicated the production of a compound with a mobility identical to progesterone. These results described the expression of the 3 beta-HSD and the activity of this metabolic enzyme in bovine oocytes and preimplantation embryos, suggesting that steroids may act as autocrine effectors on preimplantation embryo development.

11beta-Hydroxysteroid dehydrogenase types 1 and 2 are up- and downregulated in cortisol-secreting adrenal adenomas.

11beta-Hydroxysteroid dehydrogenase types 1 and 2 (11betaHSD1 and 11betaHSD2) are two isoenzymes that convert inactive glucocorticoids (e.g., cortisone) to their active forms (e.g., cortisol) and vice versa. Abundant evidence indicates that 11betaHSD2 is expressed as mRNA and protein in both adrenal cortex and adrenal tumors. In contrast, 11betaHSD1 has been investigated to a much lesser degree. We therefore studied and compared the expression and activity of the two isoenzymes in the human adrenal cortex (HAC) and cortisol-secreting adenomas (CSAs). Six HAC and six CSA specimens were studied. 11betaHSD1 and 11betaHSD2 gene expression was studied by conventional and semiquantitative reverse transcription-polymerase chain reaction. 11betaHSD1 and 11betaHSD2 activity was assayed by measuring the capacity of both microsomal fraction and tissue fragments to convert [3H]cortisone to [3H]cortisol and vice versa. Steroid hormones were separated and purified by high-performance liquid chromatography, and cortisol concentration was measured by radioimmunoassay. Semiquantitative reverse transcription-polymerase chain reaction and enzymatic assay demonstrated higher 11betaHSD1 expression and activity and lower 11betaHSD2 expression and activity in CSAs than in HACs. CSA slices secreted larger amounts of cortisol than did HAC specimens, and the cholesterol side-chain-cleaving enzyme inhibitor aminoglutethimide, by blocking the early step of steroid synthesis, reduced cortisol secretion by approximately 70%. Aminoglutethimide decreased [3H]cortisol production from [3H]cortisone and increased [3H]cortisone production from [3H]cortisol in both tissues, thereby annulling differences in 11betaHSD1 and 11betaHSD2 activity between HACs and CSAs. Collectively, our findings indicate that 1) both 11betaHSD isoenzymes are expressed as mRNA and proteins in the HAC and CSA, with 11betaHSD1 upregulated and 1betaHSD2 downregulated in CSAs; and 2) 11betaHSD1 and 11betaHSD2 activity is positively and negatively correlated with the intracellular concentration of steroid hormones. Hence, 11betaHSD isoenzymes could act as amplifiers of the secretagogue effect of agonists and could contribute to the elevated hormonal secretion of CSAs.

Absence of Cushingoid Phenotype in a Patient with Cushing’s Disease due to Defective Cortisone to Cortisol Conversion

Cushing's syndrome invariably presents with a classical phenotype comprising central adiposity, prominence of dorsal, supraclavicular and temporal fat pads, bruising, abdominal striae, proximal myopathy, and hypertension. We report the case of a 20-yr-old student with pituitary-dependent Cushing's syndrome who was spared this classical phenotype because of a defect in the peripheral conversion of cortisone to cortisol. She presented to her general practitioner with secondary amenorrhea. Clinical examination revealed normal fat distribution (body mass index, 20.9 kg/m(2)), absence of hirsutism, myopathy, or bruising; her blood pressure ranged from 115/70 to 122/82 mm Hg. She was investigated for biochemical hypercortisolemia because of a mildly elevated random circulating cortisol (serum cortisol, 661 nmol/liter). Cushing's syndrome was confirmed on the basis of repeatedly elevated urinary free cortisols (831-1049; reference range, <350 nmol/24 h), failure of low-dose dexamethasone suppression (611 nmol/liter) and loss of circadian cortisol secretion. Investigations suggested Cushing's disease; there was suppression after high-dose dexamethasone (<20 nmol/liter) and a 950% increase in ACTH after stimulation with CRH. Pituitary magnetic resonance imaging revealed a 3-mm adenoma within the pituitary gland. Urinary corticosteroid metabolites were analyzed by gas chromatography-mass spectrometry and demonstrated a decreased THF+allo-THF/THE ratio of 0.66 (mean +/- SE in Cushing's disease, 1.74 +/- 0.24) suggesting a defect in 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), an enzyme that converts the inactive glucocorticoid cortisone to active cortisol. Transphenoidal microadenomectomy was performed, and histology confirmed the diagnosis of a corticotroph adenoma. Postoperatively, serum cortisol was undetectable and replacement therapy was commenced. Subsequent investigations revealed a significantly impaired ability to convert an oral dose of cortisone acetate (25 mg) to cortisol, reduced serum cortisol to cortisone ratios, and a reduced serum half-life for cortisol (57.3 min). These results provide strong evidence for a partial defect in 11beta-HSD1 activity and concomitant increase in cortisol clearance rate. We have described a case of Cushing's disease that failed to present with a classical phenotype, and we postulate that this is due to a partial defect of 11beta-HSD1 activity, the defect in cortisone to cortisol conversion increasing cortisol clearance and thus protecting the patient from the effects of cortisol excess. This observation may help to explain individual susceptibility to the adverse effects of glucocorticoids.

Metyrapone-induced corticosterone deficiency impairs glucose oxidation and steroidogenesis in Leydig cells of adult albino rats.

The present study was designed to identify the effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis in adult male rats. Adult Wistar rats (200-250 g body weight) were treated with metyrapone, an inhibitor of corticosterone synthesis (10 mg/100 g body weight, s.c., twice daily) for 10 days. Experimental animals were killed along with controls, blood was collected, and sera separated for testosterone and estradiol assays. Testes were removed and Leydig cells were isolated, purified and used for estimating the specific activity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 14C-glucose oxidation. Serum testosterone (p < 0.05), Leydig cellular 14C-glucose oxidation (p < 0.001) and the specific activity of 17beta-HSD (p < 0.01) were significantly decreased in metyrapone treated rats. However, serum estradiol was not markedly altered compared to control. In addition to this, a set of in vitro experiments were also performed to identify the effects of metyrapone-induced corticosterone deficiency on hCG and prolactin-induced Leydig cell testosterone production. Metyrapone treatment significantly (p < 0.05) decreased the Leydig cellular basal as well as hCG and its combination with prolactin stimulated testosterone production in vitro. It is concluded from the present study that the inhibitory effects of metyrapone-induced corticosterone deficiency on Leydig cell steroidogenesis are mediated through impaired glucose oxidation and 17beta-HSD activity. In vitro studies showed that corticosterone deficiency impairs not only hCG action but also the potentiating effect of prolactin on Leydig cell steroidogenesis.

Progesterone metabolism in human leukemic monoblast U937 cells.

Progesterone markedly inhibits the functions of human macrophages and T lymphocytes, and acts as an immunosuppressant during pregnancy. It is important to examine progesterone metabolites to understand the overall bioactive properties of this sex steroid. However, progesterone metabolism has not been examined in human immune cells. The human leukemic monoblast U937 cell line exhibits monocytic lineage and provides a valuable model to analyze monocyte-macrophage differentiation. Therefore, in this study, we analyzed progesterone metabolism in U937 cells by thin-layer chromatography. Progesterone was metabolized to 5alpha-pregnan-3beta,6alpha-diol-20-one via 5alpha-dihydroprogesterone and 5alpha-pregnan-3beta-ol-20-one, and 5alpha-pregnan-3beta,20alpha-diol was also detected as a final metabolic product via 20alpha-dihydroprogesterone and 5alpha-pregnan-20alpha-ol-3-one. 5alpha-reduction (5alpha-reductase type 1) and 20alpha-reduction were involved in the first step of metabolism. To identify the enzyme responsible for the 20alpha-reduction, we screened an U937 cDNA library, and obtained a clone (1.2 kb), which was identical to the human hepatic bile acid-binding protein or 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). 293 cells transfected with this cDNA demonstrated marked 20alpha-reduction of progesterone to 20alphaDHP, but 20alpha-oxidative, 3alpha-HSD or 17beta-HSD activity was found to be negligible. In experimental animals, the importance of 20alpha-HSD has been reported to be involved in the protection of immune cells from the toxic effects of progesterone. Therefore, our present data suggest that 20alpha-HSD plays an important role in the regulation of progesterone actions in human immune cells.

Role of ketoconazole treatment in urinary-free cortisol-to-cortisone and tetrahydrocortisol-to-tetrahydrocortisone ratios in nonectopic Cushing's syndrome.

We hypothesized that in nonectopic Cushing syndrome there is an insufficient activity of type II (renal) 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) that is related to cortisol excess, rather than to corticotropin (adrenocorticotropic hormone [ACTH]) levels. We measured plasma ACTH and urinary-free cortisol (UFF), urinary-free cortisone (UFE), tetrahydrocortisol (UTHF), and tetrahydrocortisone (UTHE) in 24-h urine samples of 24 healthy subjects and 15 patients diagnosed with nonectopic Cushing syndrome. Then, in the group of patients, a new 24-h urine sample was collected after treatment with 800 mg daily of ketoconazole. The UFF/UFE and UTHF/UTHE ratios were calculated as an estimation of 11beta-HSD2 activity. The patients had an increase in both the UFF/UFE (19.95 +/- 10.3 vs 5.78 +/- 4.72 nmol/24 h; p < 0.0001) and UTHF/UTHE ratios (5.36 +/- 5.23 vs 1.39 +/- 0.95 nmol/24 h; p < 0.001). Both UFF/UFE and UTHF/UTHE ratios decreased after ketoconazole treatment (19.95 +/- 10.3 vs 12.2 +/- 6.9 nmol/24 h; p < 0.005; and 5.36 +/- 5.23 vs 1.62 vs 1.21 nmol/24 h; p < 0.001, respectively). The control subjects had a significant relationship between UFF and UFE (r = 0.70, p < 0.0001), and between UTHF and UTHE (r = 0.75, p < 0.0001) that did not exist in the patient group. After ketoconazole treatment, the decrease in cortisol excretion in the patient group allowed a positive and significant relation between UFF and UFE (r = 0.64, p < 0.01) and between UTHF and UTHE (r = 0.56, p < 0.05) to appear. There was not any significant relationship between either UFF/UFE or UTHF/UTHE ratios and plasma levels of ACTH.