Cloning of chicken 11beta-hydroxysteroid dehydrogenase type 1 and its tissue distribution

11beta-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that interconverts active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inactive 11-oxo derivatives (cortisone, 11-dehydrocorticosterone). Although bidirectional, it is considered to operate in vivo as an 11-reductase that regenerates active glucocorticoids and thus amplifies their local activity in mammals. Here we report the cloning, characterization and tissue distribution of chicken 11HSD1 (ch11HSD1). Its cDNA predicts a protein of 300 amino acids that share 51-56% sequence identity with known mammalian 11HSD1 proteins, while in contrast to most mammals, ch11HSD1 contains only one N-linked glycosylation site. Analysis of the tissue distribution pattern by RT-PCR revealed that ch11HSD1 is expressed in a large variety of tissues, with high expression in the liver, kidney and intestine, and weak in the gonads, brain and heart. 11-Reductase activity has been found in the liver, kidney, intestine and gonads with low or almost zero activity in the brain and heart. These results provide evidence for a role of 11HSD1 as a tissue-specific regulator of glucocorticoid action in non-mammalian vertebrates and may serve as a suitable model for further analysis of 11HSD1 evolution in vertebrates.     

Inhibition of 11 beta-hydroxysteroid dehydrogenase type 2 by dithiocarbamates

Dithiocarbamates (DTCs), important therapeutic and industrial chemicals released in high quantities into the environment, exhibit complex chemical and biological activities. Here, we demonstrate an effect of DTCs on glucocorticoid action due to inhibition of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 2, converting cortisol to cortisone in the kidney, but not 11 beta-HSD1, catalyzing the reverse reaction in liver and adipose tissue. Thus, DTCs may locally increase active glucocorticoid concentrations. Preincubation with the DTC thiram abolished 11 beta-HSD2 activity, suggesting irreversible enzyme inhibition. The sulfhydryl protecting reagent dithiothreitol blocked thiram-induced inhibition and NAD+ partially protected 11 beta-HSD2 activity, indicating that DTCs act at the cofactor-binding site. A 3D-model of 11 beta-HSD2 identified Cys90 in the NAD(+)-binding site as a likely target of DTCs, which was supported by a 99% reduced activity of mutant Cys90 to serine. The interference of DTCs with glucocorticoid-mediated responses suggests a cautious approach in the use of DTCs in therapeutic applications and in exposure to sources of DTCs such as cosmetics and agricultural products by pregnant women and others.     

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.     

Porcine 11beta-hydroxysteroid dehydrogenase type 2 isoform: complete coding sequence and polymorphisms

11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is involved in the regulation of the peripheral glucocorticoid concentrations. Due to the central role of glucocorticoids in protein turnover, 11beta-HSD2 is a candidate gene for optimising production traits in livestock. In addition, mutant 11beta-HSD2 animals may be used as models for human disorders. Here, we present the complete porcine 11beta-HSD2 coding sequence, the RT-PCR strategy for the examination of the coding sequence and the polymorphisms found in the pig.     

Glycyrrhizic acid suppresses type 2 11 beta-hydroxysteroid dehydrogenase expression in vivo

Licorice-derivatives such as glycyrrhizic acid (GA) competitively inhibit 11β-hydroxysteroid dehydrogenase(11β-HSD) type 2 (11-HSD2) enzymatic activity, and chronic clinical use often results in pseudoaldosteronism. Since the effect of GA on 11-HSD2 expression remains unknown, we undertook in vivo and in vitro studies. Male Wistar rats were given 30, 60 or 120 mg/kg of GA twice a day for 2 weeks. Plasma corticosterone was decreased in those given the 120 mg dose, while urinary corticosterone excretion was increased in those given the 30 and 60 mg doses but decreased in those given 120 mg GA. NAD⁺-dependent dehydrogenase activity in kidney microsomal fraction was decreased in animals receiving doses of 60 and 120 mg GA. The 11-HSD2 protein and mRNA levels were decreased in those given 120 mg GA. In contrast, in vitro studies using mouse kidney M1 cells revealed that 24 h treatment with glycyrrhetinic acid did not affect the 11-HSD2 mRNA expression levels. Thus, in addition to its role as a competitive inhibitor of 11-HSD2, the chronic high dose of GA suppresses mRNA and protein expression of 11-HSD2 possibly via indirect mechanisms. These effects may explain the prolonged symptoms after cessation of GA administration in some pseudoaldosteronism patients.     

The type I and type II 11beta-hydroxysteroid dehydrogenase enzymes.

Local tissue concentrations of glucocorticoids are modulated by the enzyme 11β-hydroxysteroid dehydrogenase which interconverts cortisol and the inactive glucocorticoid cortisone in man, and corticosterone and 11-dehydrocorticosterone in rodents. The type I isoform (11β-HSD1) is a bidirectional enzyme but acts predominantly as a oxidoreductase to form the active glucocorticoids cortisol or corticosterone, while the type II enzyme (11β-HSD2) acts unidirectionally producing inactive 11-keto metabolites. There are no known clinical conditions associated with 11β-HSD1 deficiency, but gene deletion experiments in the mouse indicate that this enzyme is important both for the maintenance of normal serum glucocorticoid levels, and in the activation of key hepatic gluconeogenic enzymes. Other important sites of action include omental fat, the ovary, brain and vasculature. Congenital defects in the 11β-HSD2 enzyme have been shown to account for the syndrome of apparent mineralocorticoid excess (AME), a low renin severe form of hypertension resulting from the overstimulation of the non-selective mineralocorticoid receptor by cortisol in the distal tubule of the kidney. Inactivation of the 11β-HSD2 gene in mice results in a phenotype with similar features to AME. In addition, these mice show high neonatal mortality associated with marked colonic distention, and remarkable hypertrophy and hyperplasia of the distal tubule epithelia. 11β-HSD2 also plays an important role in decreasing the exposure of the fetus to the high levels of maternal glucocorticoids. Recent work suggests a role for 11β-HSD2 in non-mineralocorticoid target tissues where it would modulate glucocorticoid access to the glucocorticoid receptor, in invasive breast cancer and as a mechanism providing ligand for the putative 11-dehydrocorticosterone receptor. While previous homologies between members of the SCAD superfamily have been of the order of 20–30% phylogenetic analysis of a new branch of retinol dehydrogenases indicates identities of >60% and overlapping substrate specificities. The availability of crystal structures of family members has allowed the mapping of conserved 11β-HSD domains A–D to a cleft in the protein structure (cofactor binding domain), two parallel β-sheets, and an -helix (active site), respectively.     

Piperidine amides as 11beta-hydroxysteroid dehydrogenase type 1 inhibitors

A series of piperidine amide inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) were identified via modifications of the HTS hit compound 1. The synthesis, in vitro biological evaluation, and structure-activity relationship of these compounds are presented.     

The human gene for 11 beta-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization

The Type I (mineralocorticoid) receptor has identical affinities in vitro for cortisol and aldosterone. It has been suggested that the selective role of aldosterone in regulating sodium homeostasis relies on the microsomal enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD). This enzyme converts cortisol to its inactive metabolite, cortisone, preventing cortisol from binding to the Type I receptor. We have isolated human cDNA clones encoding 11-HSD from a human testis cDNA library by hybridization with a previously isolated rat 11-HSD cDNA clone. The cDNA contains an open reading frame of 876 bases, which predicts a protein of 292 amino acids. The sequence is 77% identical at the amino acid level to rat 11-HSD cDNA. The mRNA is widely expressed, but the level of expression is highest in the liver. Hybridization of the human 11-HSD cDNA to a human-hamster hybrid cell panel localized the single corresponding HSD11 gene to chromosome 1. This gene was isolated from a chromosome 1 specific library using the cDNA as a probe. HSD11 consists of 6 exons and is at least 9 kilobases long. The data developed in this study should be applicable to the study of patients with hypertension due to apparent mineralocorticoid excess, a deficiency in 11-HSD activity.     

Guinea pig 11beta-hydroxysteroid dehydrogenase type 1: primary structure and catalytic properties

The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme is responsible for the interconversion of glucocorticoids and their inactive metabolites, and thus modulates the intracellular level of bioactive glucocorticoids. The present study was designed to clone and characterize 11beta-HSD1 in the guinea pig, a laboratory animal known for resistance to glucocorticoids. The cDNA encoding guinea pig 11beta-HSD1 was cloned by a modified 3'-RACE (rapid amplification of cDNA ends) protocol using the hepatic RNA as template. The cloned cDNA encodes a protein of 300 amino acids that shares 71 to 74% sequence identity with other known mammalian 11beta-HSD1 proteins. Sequence comparison analysis revealed that the deduced guinea pig 11beta-HSD1 was longer, by eight amino acids at the C terminus, than those of other mammals. Moreover, one of the two absolutely conserved consensus sites for N-glycosylation was absent. To examine the functional significance of these structural changes, we also characterized 11beta-HSD1 activity in the hepatic microsomes. Although the guinea pig hepatic enzyme was NADP(H)-dependent and reversible, it displayed equal affinity for cortisol and cortisone (apparent K(m) for both substrates was 3 microM). This is in marked contrast to 11beta-HSD1 in other mammals whose affinity for cortisone is approximately 10 times higher than that for cortisol (apparent K(m) of 0.3 vs. 3.0 microM). The apparent lower affinity of the guinea pig enzyme for cortisone would suggest that the intracellular bioformation of cortisol from circulating cortisone may be less efficient in this species. Northern blot analysis and RT-PCR revealed that the mRNA for 11beta-HSD1 was widely expressed in the adult guinea pig but at low amounts. In conclusion, the present study has identified distinct features in the deduced primary structure and catalytic function of 11beta-HSD1 in the guinea pig. Thus, the guinea pig provides a useful model in which the structural determinants of catalytic function of 11beta-HSD1 may be studied.     

Glucocorticoids, 11 beta-hydroxysteroid dehydrogenase type 1, and visceral obesity

Glucocorticoids are implicated as a pathophysiological mediator of obesity and its accompanying metabolic and cardiovascular complications. Obese patients exhibit normal circulating cortisol levels, related to increased glucocorticoid production and degradation. However, it has been demonstrated that local production of active cortisol from inactive cortisone driven by 11 beta-hydroxysteroid dehydrogenase type 1 is exaggerated in adipose tissue of obese subjects. Such local hypercortisolism may be responsible for increased adipocyte differentiation and enhanced secretion of free fatty acids and other substances involved in the metabolic and cardiovascular complications observed in obesity.     

Novel non-steroidal inhibitors of human 11beta-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) regulates glucocorticoid action at the pre-receptor stage by converting cortisone to cortisol. 11β-HSD1 is selectively expressed in many tissues including the liver and adipose tissue where metabolic events are important. Metabolic syndrome relates to a number of metabolic abnormalities and currently has a prevalence of >20% in adult Americans. 11β-HSD1 inhibitors are being investigated by many major pharmaceutical companies for type 2 diabetes and other abnormalities associated with metabolic syndrome. In this area of intense interest a number of structural types of 11β-HSD1 inhibitor have been identified. It is important to have an array of structural types as the physicochemical properties of the compounds will determine tissue distribution, HPA effects, and ultimately clinical utility. Here we report the discovery and synthesis of three structurally different series of novel 11β-HSD1 inhibitors that inhibit human 11β-HSD1 in the low micromolar range. Docking studies with 1–3 into the crystal structure of human 11β-HSD1 reveal how the molecules may interact with the enzyme and cofactor and give further scope for structure based drug design in the optimisation of these series.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Oxazolones as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

2,5,5-Trisubstituted oxazolones were identified as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The synthesis, structure-activity relationship and metabolic stability of these compounds are presented.     

11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue and prospective changes in body weight and insulin resistance

Objective: Increased mRNA and activity levels of 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in human adipose tissue (AT) are associated with obesity and insulin resistance. The aim of our study was to investigate whether 11βHSD1 expression or activity in abdominal subcutaneous AT of non‐diabetic subjects are associated with subsequent changes in body weight and insulin resistance [homeostasis model assessment of insulin resistance (HOMA‐IR)]. Research Methods and Procedures: Prospective analyses were performed in 20 subjects (two whites and 18 Pima Indians) who had baseline measurements of 11βHSD1 mRNA and activity in whole AT (follow‐up, 0.3 to 4.9 years) and in 47 Pima Indians who had baseline assessments of 11βHSD1 mRNA in isolated adipocytes (follow‐up, 0.8 to 5.3 years). Results: In whole AT, although 11βHSD1 mRNA levels showed positive associations with changes in weight and HOMA‐IR, 11βHSD1 activity was associated with changes in HOMA‐IR but not in body weight. 11βHSD1 mRNA levels in isolated adipocytes were not associated with follow‐up changes in any of the anthropometric or metabolic variables. Discussion: Our results indicate that increased expression of 11βHSD1 in subcutaneous abdominal AT may contribute to risk of worsening obesity and insulin resistance. This prospective relationship does not seem to be mediated by increased 11βHSD1 expression in adipocytes.     

Human adipose tissue under in vitro inhibition of 11beta-hydroxysteroid dehydrogenase type 1: differentiation and metabolism changes.

In humans, oxoreducing 11beta-HSD-1 activity appears to be related to body fat distribution in male-type central obesity, but not in female-type peripheral obesity. We postulated that inhibition of 11beta-HSD-1 might have clinical therapeutic significance in oxoreducing mostly visceral fat and its metabolic activity. Our current study investigated the consequence at the cellular level of such inhibition. As an inhibitor of 11beta-HSD-1 activity, we used the licorice derivative carbenoxolone. Carbenoxolone has an inhibitory effect on the activity of both oxidizing 11beta-HSD-2, which converts cortisol to cortisone, and oxoreducing 11beta-HSD-1; yet, preadipocytes and adipocytes only express the latter. Preadipocytes were retrieved from omental and subcutaneous fat from healthy non-obese individuals and differentiated in vitro to mature adipocytes. Activity of 11beta-HSD-1 was assayed by measuring conversion of added 500 nM cortisone to cortisol. Expression of 11beta-HSD-1 mRNA was determined by real-time PCR, while lipolytic effects were determined by measuring glycerol and triglyceride concentration in the culture medium. Carbenoxolone decreased 11beta-HSD-1 activity in a dose-dependent manner with an IC-50 of 5X10 -6 M, but did not affect the expression of 11beta-HSD-1 mRNA. Cortisone stimulated subcutaneous, but not omental preadipocytes proliferation, an effect that was not abolished by carbenoxolone. Dexamethasone had a stimulatory effect on the maturation of both omental and subcutaneous preadipocytes. Carbenoxolone per se, either with or without cortisone, had a negative effect on preadipocyte maturation. Inhibiting 11beta-HSD-1 activity by carbenoxolone had no impact on leptin secretion. Thus, carbenoxolone has no effect on preadipocyte proliferation, but a dramatic inhibitory effect on preadipocyte differentiation into mature adipocytes. The mechanism is only partly related to its inhibitory effect on 11beta-HSD-1 activity. The present observations lend support to the presence of an intracrine loop of a hormone that is both produced from a precursor and active within the preadipocyte and adipocyte.     

Luteinizing hormone induces expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells

Background  

Leydig cells are the primary source of testosterone in male vertebrates. The biosynthesis of testosterone in Leydig cells is strictly dependent on luteinizing hormone (LH). On the other hand, it can be directly inhibited by excessive glucocorticoid (Corticosterone, CORT, in rats) which is beyond the protective capability of 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and type 2 (11beta-HSD2; encoded by gene Hsd11b2 in rats) in Leydig cells. Our previous study found that LH increases 11beta-HSD1 expression in rat Leydig cells, but the effect of LH on the expression and activity of 11beta-HSD2 is not investigated yet.     

Interactions between 11beta-hydroxysteroid dehydrogenase and COX-2 in kidney

The syndrome of apparent mineralocorticoid excess (SAME) is an autosomal recessive form of salt-sensitive hypertension caused by deficiency of the kidney type 2 11beta-hydroxysteroid dehydrogenase (11betaHSD2). In this disorder, cortisol is not inactivated by 11betaHSD2, occupies mineralocorticoid receptors (MRs), and causes excessive sodium retention and hypertension. In renal medulla, prostaglandins derived from cyclooxygenase-2 (COX-2) stimulate sodium and water excretion, and renal medullary COX-2 expression increases after mineralocorticoid administration. We investigated whether medullary COX-2 also increases in rats with 11betaHSD2 inhibition and examined its possible role in the development of hypertension. 11betaHSD2 inhibition increased medullary and decreased cortical COX-2 expression in adult rats and induced high blood pressure in high-salt-treated rats. COX-2 inhibition had no effect on blood pressure in control animals but further increased blood pressure in high-salt-treated rats with 11betaHSD2 inhibition. COX-1 inhibition had no effect on blood pressure in either control or experimental animals. 11betaHSD2 inhibition also led to medullary COX-2 increase and cortical COX-2 decrease in weaning rats, primarily through activation of MRs. In the suckling rats, medullary COX-2 expression was very low, consistent with a urinary concentrating defect. 11betaHSD2 inhibition had no effect on either cortical or medullary COX-2 expression in the suckling rats, consistent with low levels of circulating corticosterone in these animals. These data indicate that COX-2 plays a modulating role in the development of hypertension due to 11betaHSD2 deficiency and that 11betaHSD2 regulates renal COX-2 expression by preventing glucocorticoid access to MRs during postnatal development.