beta-HSD Activity Research Articles

Reduced activity of 11β‐hydroxysteroid dehydrogenase type 2 is not responsible for sodium retention in nephrotic rats

In mineralocorticoid target cells 11-beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) converts glucocorticoids into non-active metabolites thereby protecting the mineralocorticoid receptor (MR) from stimulation by glucocorticoids. In nephrotic syndrome, a decreased activity of 11betaHSD2 has been suggested to allow glucocorticoids to stimulate MR, thereby contributing to sodium retention. We tested this hypothesis in the puromycin aminonucleoside model of nephrotic syndrome in rats. Complete sodium and potassium intakes and excretions (faeces and urine) were measured in rats in metabolic cages. RNase protection assay of mRNA and Western blotting of protein were used to estimate renocortical expression of 11betaHSD2 and of the MR downstream effector serum and glucocorticoid induced kinase (SGK). In an intervention series, dexamethasone was given [10 microg (100 g bw)(-1)] to suppress endogenous glucocorticoids in the proteinuric stage during active sodium retention. Nephrotic rats developed proteinuria, positive sodium balance, decreased plasma aldosterone concentration, and decreased urinary Na(+)/K(+) ratio. 11betaHSD2 mRNA expression was down-regulated but protein expression was unchanged. SGK mRNA and phosphorylated SGK protein were up-regulated while total SGK protein expression was unchanged. Dexamethasone treatment, which suppressed plasma corticosterone concentration, did not correct sodium balance or fluid retention in nephrotic rats. Our results do not support the hypothesis that stimulation of the MR by endogenous glucocorticoids induces sodium and fluid retention in experimental nephrotic syndrome in rats.

Salt-sensitive blood pressure—an intermediate phenotype predisposing to diabetic nephropathy?

Family studies point to important genetic determinants of diabetic nephropathy (DN). Blood pressure (BP) is higher in offspring of patients with type 2 diabetes and DN, but the pathomechanisms involved have not been elucidated. We examined the salt sensitivity of BP after 5 days equilibration on a low (20 mmol/day) vs high salt diet (220 mmol/day) in three matched groups of 15 subjects each: (i) control individuals; (ii) offspring of type 2 diabetic parents without DN (DN-); and (iii) offspring of type 2 diabetic parents with DN (DN+). Ambulatory BP and hormones involved in sodium homeostasis [plasma renin activity (PRA), aldosterone and atrial natriuretic peptide (ANP)] as well as the tetrahydrocortisol + 5-allotetrahydrocortisol/tetrahydrocortisone (THF + 5alphaTHF)/THE) ratio in the urine as an index of 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) activity were analysed. In offspring of DN+ patients on a high salt diet, systolic and diastolic BP was 137/82+/-10/8 mmHg vs 125/77+/-12/8 mmHg in offspring of DN- patients (P<0.01 for systolic BP). The salt-induced difference in mean BP between high and low salt diet was 5.2+/-3.3 mmHg in offspring of DN+ patients vs 0.7+/-4.7 mmHg in offspring of DN- patients (P<0.002). The proportion of 'salt-sensitive' individuals was 67% in offspring of DN+ patients vs 20% in offspring of DN- patients (P<0.05). In all groups, a high salt diet caused a comparable decrease of PRA and p-aldosterone accompanied by an increase in ANP. The urinary (THF + 5alphaTHF)/THE ratio was 1.23+/-0.36 in salt-sensitive individuals and 0.99+/-0.33 (P<0.03) in salt-resistant subjects, consistent with increased activity of 11betaHSD2. BP is more salt sensitive in offspring of type 2 diabetic patients with diabetic nephropathy. The salt sensitivity of BP may be an intermediate phenotype in individuals with a high risk of future diabetic nephropathy.

The Effects of Chorioamnionitis and Betamethasone on 11β, Hydroxysteroid Dehydrogenase Types 1 and 2 and the Glucocorticoid Receptor in Preterm Human Placenta

Preterm birth is one of the major problems faced in perinatal medicine and is often associated with underlying clinical infection. Treatment with maternal betamethasone has helped to improve neonatal morbidity and mortality. We hypothesized that betamethasone treatment and chorioamnionitis would alter the bioavailability of placental glucocorticoids through the regulation of the 11beta hydroxysteroid dehydrogenase (11beta HSD) isozymes and the glucocorticoid receptor (GR). Placental samples were obtained from three groups of women who delivered prematurely: (1) those who delivered in the absence of infection, (2) those who received betamethasone treatment before delivering without infection, and (3) those who had pregnancies complicated with chorioamnionitis. Western blotting was used to determine 11beta HSD-1, 11beta HSD-2, GRT, and GRalpha expression, and 11beta HSD-2 activity was determined in each group. JEG-3 cells were used to study the regulation of the 11beta HSD isozymes. In cases of chorioamnionitis where mothers had not been treated with betamethasone, placental 32-kd 11beta HSD-1 protein expression was increased. In cases of chorioamnionitis regardless of betamethasone treatment, placental 11beta HSD-2 expression and activity was decreased compared to controls. In these placental samples, the expression of GRT and GRalpha did not change significantly. In JEG-3 cells, 11beta HSD-1 32-kd expression was increased with interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), while 11beta HSD-2 expression was unaffected. These data suggest that there could be an increased fetal exposure to maternal glucocorticoids in cases of chorioamnionitis as a result of changes in the expression and activity of the placental 11beta HSD isozymes.

Seasonal Changes in the Activity of 11β‐Hydroxysteroid Dehydrogenase in Toad Testes

In mammals, glucocorticoids (GC) are inactivated by the oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). To study that mechanism in the testes of Bufo arenarum, 11beta-HSD activity and plasma corticosterone (B) were determined in toads from pre- (PR), post- (P), and reproductive (R) periods. Toad 11beta-HSD is NAD(+)-dependent and strongly inhibited by glycyrrhetinic acid. V(max) is higher in the P period (1.37 vs. R: 0.13 and PR: 0.26 pmol/min-mg protein), whereas K(m) values (around 200 nM) remain constant. Plasma B concentrations (R: 178; PR: 61; P: 114 nM) are close to K(m) values, suggesting that, under baseline conditions, 11beta-HSD could protect testes against GC action.

Dosagem de cortisol e cortisona livres urinários empregando cromatografia líquida associada a espectrometria de massa em tandem (LC-MS/MS)

Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation, based on automated preparative methods and tandem mass spectrometry, described in our country.

11β-Hydroxysteroid Dehydrogenase 2 in Rat Leydig Cells: Its Role in Blunting Glucocorticoid Action at Physiological Levels of Substrate

Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.

The transplacental passage of prednisolone in pregnancies complicated by early-onset HELLP syndrome

During pregnancy the placental 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) enzyme inactivates prednisolone by interconversion into prednisone, protecting the fetus from high levels of prednisolone. Recent reports suggest decreased placental 11beta-HSD2 activity in pregnancies complicated by preeclampsia. The purpose of our investigation was to study the transplacental passage of prednisolone in patients suffering from early preterm HELLP syndrome, a severe complication of preeclampsia. We examined the maternal and umbilical cord plasma concentration of prednisolone in nine women receiving 50 mg of prednisolone twice a day. Samples were obtained during caesarean section at a gestational age between 27 and 31 weeks. Mean fetal concentration was 10-fold lower as compared to maternal prednisolone concentration (mean+/-SD 52.8 nmol/L+/-27.0 vs. 477.5 nmol/L+/-300, p<0.01). A significant correlation was found between the last dose of prednisolone to delivery interval and the fetal prednisone concentration (Spearman's correlation coefficient r=-0.946, p<0.000). Our data demonstrate unimpaired placental 11beta-HSD2 activity in patients suffering from HELLP syndrome at early gestational age as shown by both a 10-fold lower fetal prednisolone concentration as compared to the mother and a strong correlation between the last dose of prednisolone to delivery interval and the fetal prednisone concentration. Prednisolone may therefore have less effect on the fetus than betamethasone or dexamethasone.

Histochemical localization of enzymes and lipids in the ovary of a vespertilionid bat, Scotophilus heathi, during the reproductive cycle

The present study describes seasonal changes in delta5 3beta hydroxysteroid dehydrogenase (3beta-HSD), glusose-6 phosphates dehydrogenase (G-6-PD), and lipids in the ovary of a vespertilionid bat, Scotophilus heathi. Total lipids and 3beta-HSD activity are restricted to thecal and interstitial cells of the ovary. The total lipids, 3beta-HSD, and G-6-PD significantly increase during recrudescence, and remain high during winter dormancy and breeding as compared to the other reproductive phases. High incidence of lipids and enzyme activity in interstitial cells during the breeding period and at the time of ovulation clearly suggests that these cells are actively involved in steroidogenesis. A decline in enzymes and lipid activity during winter dormancy, which correlates with the declining levels of steroidogenesis, might be the factors responsible for prolonged survival of the Graafian follicle in the ovary of S. heathi.

Tumor necrosis factor alpha and phorbol 12‐myristate‐13‐acetate down‐regulate human 11β‐hydroxysteroid dehydrogenase type 2 through p50/p50 NF‐κB homodimers and Egr‐1

The 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) regulates access of 11beta-hydroxyglucocorticoids to the mineralocorticoid receptor by reducing the hydroxyl group of these steroids at position 11. Previous cell culture studies revealed that tumor necrosis factor-alpha (TNF-alpha) down-regulates 11beta-HSD2 activity. Here, we demonstrate that transgenic mice overexpressing TNF-alpha have decreased mRNA abundance and activity of 11beta-HSD2 in kidney tissue, indicating that this effect may occur also in vivo. The analysis of the transcriptional regulation of 11beta-HSD2 by TNF-alpha and phorbol 12-myristate-13-acetate (PMA) with in vivo genomic footprinting in human colon SW620 cells revealed stimulus-dependent protein-DNA interactions in three promoter regions, kappaB1, Sp1/Egr-1I, and Sp1/Egr-1II. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated the relevance of NF-kappaB binding to kappaB1 and of Egr-1 binding to Sp1/Egr-1 sites for the PMA and TNF-alpha effect. We observed a temporal switch of binding to kappaB1 site from active p65/p50 heterodimers to inactive p50/p50 homodimers. TNF-alpha or PMA treatment for 24 h resulted in accumulation of p50 and decrease of p65 nuclear proteins. Overexpression of p50 inhibited HSD11B2 promoter activity and overexpression of Egr-1 inhibited transactivation of the HSD11B2 promoter by p65/p50. In conclusion, TNF-alpha and PMA down-regulate expression and activity of 11beta-HSD2 most likely by a coordinate binding of p50/p50 and Egr-1 to the HSD11B2 promoter.

Molecular Biology of the 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase Gene Family

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

Inflammatory Mediators Down-Regulate 11.BETA.-Hydroxysteroid Dehydrogenase Type 2 in a Human Lung Epithelial Cell Line BEAS-2B and the Rat Lung

In the lung, anti-inflammatory actions of glucocorticoids would be determined by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the microsomal enzyme responsible for the breakdown of bio-active glucocorticoids. However, regulation of 11beta-HSD2 under inflammatory conditions such as acute lung injury is not well understood. In the present study, we examined whether inflammatory substances would influence the activity and mRNA expression of 11beta-HSD2 in the lung. In a human bronchial epithelial cell line BEAS-2B, endotoxin inhibited 11beta-HSD2 enzyme activity in a dose-dependent manner over 48 h with a significant decrease in the mRNA expression. Likewise, tumor necrosis factor (TNF)-alpha inhibited both activity and mRNA expression of 11beta-HSD2. The TNF-alpha-dependent decrease in the enzyme activity was completely blocked by anti-TNF-alpha antibody, while antibody alone showed no significant influence on the enzyme activity. An nitric oxide donor (NO) sodium nitropusside or a cGMP analog 8-br-cGMP caused moderate but significant decreases in both activity and mRNA expression of 11beta-HSD2. Importantly, treatment of rats with endotoxin significantly decreased both activity and mRNA expression of 11beta-HSD2 in the lung tissue. We conclude that lung inflammation reduces local glucocorticoid breakdown and augments glucocorticoid action in the lung by down-regulating 11beta-HSD2 via multiple mechanisms.

Estradiol induced inhibition of 11β-hydroxysteroid dehydrogenase 1: an explanation for the postmenopausal hormone replacement therapy effects

The adverse and beneficial effects of postmenopausal hormone replacement therapy include: ischemic heart disease, stroke, pulmonary embolism, breast cancer, an increased rate of onset of asthma as well as reductions in the incidence of diabetes in women with known coronary artery disease and osteoporotic fractures. These varied effects can be explained by the down regulation of 11beta-hydroxysteroid dehydrogenase by estradiol, which results in a reduction of tissue specific cortisol production. The reduction in local cortisol production which diminishes the endogenous anti-inflammatory effects, also allows for the progression of both vascular and pulmonary inflammation. The decrease in cortisol activation reduces insulin resistance and anti-proliferative effects thereby reducing the potential for diabetes but allowing for the emergence of malignancy. Furthermore, the decreased local tissue availability of cortisol reduces the tendency for the development of osteoporosis. New techniques and drugs are being developed to evaluate the modulation of 11beta-HSD1 activity. Further study should result in new ways to control both inflammation and metabolism.

11β-Hydroxysteroid dehydrogenase type 2 activity is associated with left ventricular mass in essential hypertension

Left ventricular mass (LVM) is under the control of aldosterone and angiotensin II in experimental hypertension, but the effect of aldosterone on LVM is controversial in essential hypertension (EH). Some EH patients show a mild impairment of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity without clinical features of the syndrome of apparent mineralocorticoid excess, where the incomplete cortisol-to-cortisone conversion leads to glucocorticoid-mediated mineralocorticoid effects. The mineralocorticoid receptor and 11beta-HSD2 are co-expressed in human heart. We investigated whether LVM may be regulated by glucocorticoids in EH patients. The ratio between 24 h urinary tetrahydro derivatives of cortisol and cortisone (THFs/THE), plasma renin activity, 24 h urinary aldosterone, blood pressure, and LVM indexed for height(2.7) (LVMh(2.7)) were analysed in 493 never-treated hypertensives and 98 normotensives. THFs/THE was associated with LVMh(2.7) in hypertensives and normotensives (r=0.32, P<0.001, and r=0.17, P=0.04, respectively) and persisted after adjusting for confounders (multiple regression analysis). Body mass index, sex, recumbent plasma renin activity, and THFs/THE accounted for 26.1% of LVMh(2.7) variation. Urinary aldosterone was not correlated with LVMh(2.7). We suggest that glucocorticoids may take part in the regulation of LVM in EH patients as a function of 11beta-HSD2 activity, and contribute to the target organ damage associated with essential hypertension.

Regulation of 11β‐hydroxysteroid dehydrogenase type 1 and glucose‐stimulated insulin secretion in pancreatic islets of Langerhans

In rodents, the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inactive 11-dehydrocorticosterone (DHC) into active corticosterone. The mRNA and activity of 11beta-HSD1 have been shown to be present in batch-incubated pancreatic islets from the ob/ob mouse. In other tissues, 11beta-HSD1 expression has been demonstrated to be regulated by glucocorticoids. In the present study, the influence of DHC on 11beta-HSD1 levels and glucose-induced changes in insulin secretion were studied in pancreatic islets isolated from the ob/ob mouse. Western blotting with antiserum for 11beta-HSD1 verified the presence of 11beta-HSD1 in islets from obese ob/ob and normal C57BL/6J mice. Insulin secretion was determined by perifusing islets and assaying the perifusate with ELISA. Islets from the ob/ob mouse contained almost twofold more 11beta-HSD1 protein than islets from the C57BL/6J mouse. When islets from ob/ob mice were cultured with 50 nM DHC, the 11beta-HSD1 levels doubled compared with islets cultured in the absence of DHC. Selective inhibition of 11beta-HSD1 attenuated DHC-induced increase in 11beta-HSD1 levels, as did an antagonist of the glucocorticoid receptor. In individually perifused ob/ob mouse islets, early and late phases of glucose-stimulated insulin secretion (GSIS) were dose-dependently inhibited by 5, 50 and 500 nM DHC. Whereas inclusion of 11beta-HSD1 inhibitors restored, addition of the glucocorticoid receptor antagonist attenuated the DHC-mediated inhibition of GSIS. Levels of 11beta-HSD1 in islets from ob/ob mice are positively regulated by DHC and could be lowered by a selective 11beta-HSD1 inhibitor and a glucocorticoid receptor antagonist. Increased levels of 11beta-HSD1 were associated with impaired GSIS.

Glucocorticoids Stimulate the Expression of 11β-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Placental Trophoblast Cells

Proper glucocorticoid exposure in utero is vital for normal fetal organ growth and maturation. The placental enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), plays a pivotal role in controlling fetal exposure to high levels of maternal glucocorticoid by converting cortisol into its inactive metabolite, cortisone. The present study was designed to determine whether glucocorticoids auto-regulate 11beta-HSD2 in the human placenta using cultured trophoblast cells as a model system. Trophoblasts were isolated from uncomplicated term placentas and treated with glucocorticoids. The synthetic glucocorticoid dexamethasone increased 11beta-HSD2 activity in a time- and concentration-dependent manner; this effect was accompanied by a corresponding increase in 11beta-HSD2 mRNA. Furthermore, the glucocorticoid receptor antagonist, RU-486, abrogated the dexamethasone-induced increase in 11beta-HSD2 activity, suggesting that the effect of dexamethasone is mediated through the glucocorticoid receptor. Results from transient transfection and mRNA decay experiments indicate that the glucocorticoid-induced increase in 11beta-HSD2 expression is mediated at both the transcriptional and posttranscriptional levels. In conclusion, the present study demonstrates that in cultured human trophoblasts, 11beta-HSD2 is subject to auto-regulation by glucocorticoids. If this occurs in the human placenta in vivo, the glucocorticoid-induced up-regulation of placental 11beta-HSD2 would represent an important safeguard mechanism by which the fetus may be protected from detrimental exposure to elevated levels of maternal glucocorticoids.

Prenatal glucocorticoids and long-term programming

Epidemiological evidence suggests that low birth weight is associated with an increased risk of cardiovascular, metabolic and neuroendocrine disorders in adult life. Glucocorticoid administration during pregnancy reduces offspring birth weight and alters the maturation of the lung and other organs. We hypothesised that prenatal exposure to excess glucocorticoids or stress might represent a mechanism linking foetal growth with adult pathophysiology. In rats, birth weight is reduced following prenatal exposure to the synthetic steroid dexamethasone, which readily crosses the placenta, or to carbenoxolone, which inhibits 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the physiological feto-placental 'barrier' to maternal glucocorticoids. As adults, the offspring exhibit permanent hypertension, hyperglycaemic, increased hypothalamic-pituitary-adrenal (HPA) axis activity and behaviour reminiscent of anxiety. Physiological variations in placental 11beta-HSD2 activity correlate directly with foetal weight. In humans, 11beta-HSD2 gene mutations cause low birth weight. Moreover, low-birth-weight babies have higher plasma cortisol levels throughout adult life, indicating HPA axis programming. The molecular mechanisms may reflect permanent changes in the expression of specific transcription factors, key among which is the glucocorticoid receptor (GR) itself. The differential programming of the GR in different tissues reflects effects upon one or more of the multiple tissue-specific alternate first exons/promoters of the GR gene. Overall, the data suggest that both pharmacological and physiological exposure prenatally to excess glucocorticoids programmes cardiovascular, metabolic and neuroendocrine disorders in adult life.

Apparent mineralocorticoid excess syndrome: an overview

Apparent mineralocorticoid excess (AME) syndrome results from defective 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). This enzyme is co-expressed with the mineralocorticoid receptor (MR) in the kidney and converts cortisol (F) to its inactive metabolite cortisone (E). Its deficiency allows the unmetabolized cortisol to bind to the MR inducing sodium retention, hypokalemia, suppression of PRA and hypertension. Mutations in the gene encoding 11beta-HSD2 account for the inherited form, but a similar clinical picture to AME occurs following the ingestion of bioflavonoids, licorice and carbenoxolone, which are competitive inhibitors of 11beta-HSD2. Reduced 11beta-HSD2 activity may explain the increased sodium retention in preeclampsia, renal disease and liver cirrhosis. Relative deficiency of 11beta-HSD2 activity can occur in Cushing's syndrome due to saturation of the enzyme and explains the mineralocorticoid excess state that characterizes ectopic ACTH syndrome. Reduced placental 11beta-HSD2 expression might explain the link between reduced birth weight and adult hypertension. Polymorphic variability in the HSD11B2 gene in part determines salt sensitivity, a forerunner for adult hypertension onset. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2; although AME is a genetic disorder, several exogenous compounds can bring about the symptoms by inhibiting 11beta-HSD2 enzyme. Substrate excess as seen in Cushing's syndrome and ACTH ectopic production can overwhelm the capacity of 11beta-HSD2 to convert F to E, leading up to an acquired form of AME.

11beta-hydroxysteroid dehydrogenase type 1 activity in lean and obese males with type 2 diabetes mellitus.

Glucocorticoids play an important role in the pathogenesis of obesity and insulin resistance. Impaired conversion of cortisone (E) to cortisol (F) by the type 1 isoenzyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in obesity may represent a protective mechanism preventing ongoing weight gain and glucose intolerance. We have studied glucocorticoid metabolism in 33 male subjects with type 2 diabetes mellitus [age, 44.2 +/- 13 yr; body mass index (BMI), 31.1 +/- 7.5 kg/m(2) (mean +/- sd)] and 38 normal controls (age, 41.4 +/- 14 yr; BMI, 38.2 +/- 12.8 kg/m(2)). Circulating F:E ratios were elevated in the diabetic group and correlated with serum cholesterol and homeostasis model assessment-S. There was no difference in 11beta-HSD1 activity between diabetic subjects and controls. In addition, 11beta-HSD1 activity was unaffected by BMI in diabetic subjects. However, in control subjects, increasing BMI was associated with a reduction in the urinary tetrahydrocortisol+5alpha-tetrahydrocortisol:tetrahydrocortisone ratio (P < 0.05) indicative of impaired 11beta-HSD1 activity. The degree of inhibition correlated tightly with visceral fat mass. Changes in 11beta-HSD1 activity could not be explained by circulating levels of adipocytokines. Impaired E to F metabolism in obesity may help preserve insulin sensitivity and prevent diabetes mellitus. Failure to down-regulate 11beta-HSD1 activity in patients with diabetes may potentiate dyslipidemia, insulin resistance, and obesity. Inhibition of 11beta-HSD1 may therefore represent a therapeutic strategy in patients with type 2 diabetes mellitus and obesity.

Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase.

Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent. These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

11beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone stimulates chorionic gonadotropin secretion from human term cytotrophoblast cells differentiated in vitro.

To investigate the effect of altering local glucocorticoid concentration on human chorionic gonadotropin (hCG) production by cultured placental trophoblast cells. Human placental trophoblasts were isolated from fresh placentas. Cytotrophoblasts were purified and placed into 24-well multiplates. For cultivation Dulbecco's modified Eagle's medium (DMEM) with 15 mm HEPES and 15% FBS was used. 11beta-Hydroxysteroid dehydrogenase (11beta-HSD) activity and its inhibition by carbenoxolone (CE) were measured in cultured cells. Cultures were exposed to CE for 16-20 hr. Overnight production of hCG was measured by radioimmunoassay in control and treated cells. The 11beta-HSD activity in these cultures was inhibited by nm concentrations of CE, the apparent Ki being 2.5 nm. Inhibition of 11beta-HSD activity with 0.1 nm CE resulted in 1.5-fold increase in the production of hCG. Increasing local glucocorticoid concentration by the inhibition of 11beta-HSD results in higher hCG secretion, which in turn enhance cell differentiation.