beta-HSD Activity Research Articles

Rational design of novel mutants of fungal 17β-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.

Local Amplification of Glucocorticoids by 11β‐Hydroxysteroid Dehydrogenase Type 1 and Its Role in the Inflammatory Response

Glucocorticoids are widely used to treat chronic inflammatory conditions including rheumatoid arthritis. They promote mechanisms important for normal resolution of inflammation, notably macrophage phagocytosis of leukocytes undergoing apoptosis. Prereceptor metabolism of glucocorticoids by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) amplifies intracellular levels of glucocorticoids by oxoreduction of intrinsically inert cortisone (in humans, 11-dehydrocorticosterone in mice) into active cortisol (corticosterone in mice) within cells expressing the enzyme. Recently, we have shown in a mouse model of acute inflammation, high expression of 11beta-HSD oxoreductase but not dehydrogenase activity in cells elicited rapidly in the peritoneum by a single thioglycollate injection. 11beta-HSD oxoreductase activity remained high in peritoneal cells until the inflammation resolved. In vitro, the 11beta-HSD1 substrate, 11-dehydrocorticosterone, increased macrophage phagocytosis of apoptotic neutrophils to the same extent as corticosterone. This effect was dependent upon 11beta-HSD1: these cells solely expressed the type 1 11beta-HSD isozyme (not 11beta-HSD2), and carbenoxolone, an 11beta-HSD inhibitor, prevented the increase in phagocytosis elicited by 11-dehydrocorticosterone. Macrophages from 11beta-HSD1-deficient mice failed to respond to 11-dehydrocorticosterone. In vivo, 11beta-HSD1-deficient mice showed a delay in acquisition of macrophage phagocytic competence and had an increased number of free apoptotic neutrophils during sterile peritonitis. Importantly, in preliminary experiments, 11beta-HSD1-deficient mice exhibited delayed resolution of inflammation in experimental arthritis. These findings suggest 11beta-HSD1 may be a component of mechanisms engaged early during the inflammatory response that promote its subsequent resolution.

11β‐Hydroxysteroid Dehydrogenase Type 1 in Adipose Tissue and Prospective Changes in Body Weight and Insulin Resistance

Increased mRNA and activity levels of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) in human adipose tissue (AT) are associated with obesity and insulin resistance. The aim of our study was to investigate whether 11betaHSD1 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)]. Prospective analyses were performed in 20 subjects (two whites and 18 Pima Indians) who had baseline measurements of 11betaHSD1 mRNA and activity in whole AT (follow-up, 0.3 to 4.9 years) and in 47 Pima Indians who had baseline assessments of 11betaHSD1 mRNA in isolated adipocytes (follow-up, 0.8 to 5.3 years). In whole AT, although 11betaHSD1 mRNA levels showed positive associations with changes in weight and HOMA-IR, 11betaHSD1 activity was associated with changes in HOMA-IR but not in body weight. 11betaHSD1 mRNA levels in isolated adipocytes were not associated with follow-up changes in any of the anthropometric or metabolic variables. Our results indicate that increased expression of 11betaHSD1 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 11betaHSD1 expression in adipocytes.

Type 1 11 β-hydroxysteroid Dehydrogenase as Universal Drug Target in Metabolic Diseases?

Glucocorticoid hormones play essential roles in adaptation to stress, regulation of metabolism and inflammatory responses. Their effects primarily depend on their binding to intracellular receptors leading to altered target gene transcription as well as on cell-type specific biotransformation between 11beta-hydroxy glucocorticoids and their 11-oxo metabolites. The latter effect is accomplished by two different 11beta-hydroxysteroid dehydrogenase isozymes, constituting a shuttle system between the receptor ligand cortisol and its non-binding precursor cortisone. Whereas the type 1 enzyme (11beta-HSD1) is in vitro a NADP(H)- dependent bidirectional enzyme, it reduces in most instances in vivo cortisone to active cortisol. The type 2 enzyme is an exclusive NAD+ dependent dehydrogenase of glucocorticoids, thus "protecting" the mineralocorticoid receptor against illicit occupation by cortisol. Inhibition of tissue-specific glucocorticoid activation by 11beta-HSD1 constitutes a promising target in the treatment of metabolic and cardiovascular diseases. Pharmacological inhibition leads in animal models to lowered hepatic glucose production and increased insulin sensitivity, the primary goals in therapy of diabetes mellitus. Importantly, 11beta-HSD1 activity appears to be intrinsically linked to all features of the metabolic syndrome, which could at least in animal experiments be modulated by use of synthetic selective inhibitors. Importantly, these features include not only insulin resistance but also dyslipidemia, obesity and arterial hypertension. Animal studies and pharmacological experiments suggest further unrelated target areas, for example improvement of cognitive function and treatment of glaucoma, due to the role of glucocorticoids and cellular activation by 11beta-HSD1 in these pathologies. The recent development of specific 11beta-HSD1 inhibitors coupled with advances on structural knowledge and regulation of the 11beta-HSD1 target has undoubtedly promoted the understanding of glucocorticoid control of metabolic regulation. Taken together, it appears that inhibitors against 11beta-HSD1 constitute a promising avenue for novel treatment strategies against the underlying causes of cardiovascular and other metabolic diseases.

11β‐Hydroxysteroid Dehydrogenase Activity in Pregnancies Complicated by Hydatidiform Mole

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) plays an important role in regulating active glucocorticoid reaching the fetus. In normal pregnancy, placental 11beta-HSD functions primarily in oxidative direction. Placental tissue of patients with pregnancies complicated by pre-eclampsia exhibit significantly lower type 1 and 2 11beta-HSD activities and significantly high cortisol level in cord blood suggesting fetal exposure to higher level of active glucocorticoids. The activity of 11beta-HSD in gestational trophoblastic disease has not been determined. The objective of this study was to assess 11beta-HSD activity in tissue from normal second trimester and pregnancies complicated by hydatidiform mole. Normal placental tissues were obtained from patients undergoing termination of pregnancy, and from patients undergoing uterine evacuation for hydatidiform mole. Both nicotinamide adenine dinucleotide (NAD)- and nicotinamide adenine dinucleotide phosphate (NADP)-dependent activities were assayed in central villous tissue. Comparison of groups was performed using Student's t-test. A P-value of 0.05 was considered significant. Data are presented as mean +/- S.D. Tissue obtained from five patients with pathology-proven hydatidiform mole demonstrated significantly lower 11beta-HSD activities compared with placental tissue obtained from normal pregnancies. The mean NAD-dependent 11beta-HSD activity in normal placentas was 386 +/- 109 pmol/min/g placenta and in hydatidiform mole was 74 +/- 54 pmol/min/g placenta (P < 0.01). The mean NADP-dependent 11beta-HSD activity in normal placentas was 370 +/- 120 pmol/min/g placenta and in trophoblastic disease was 68 +/- 69 pmol/min/g placenta (P < 0.01). Our data indicate significant impairment in the ability of hydatidiform mole tissue to inactivate glucocorticoids.

Exaggerated adrenarche and altered cortisol metabolism in Type 1 diabetic children

Reported literature data strongly suggest that steroid metabolism is dysregulated in Type 1 diabetes mellitus. The aim of this study was to non-invasively examine the cortisol metabolism in children with Type 1 diabetes mellitus (T1DM) in detail and to test the hypothesis that adrenarche is affected under conventional intensive insulin therapy. In 24-h urine samples of 109 patients aged 4–18 years with T1DM of more than 1 year, steroids were profiled using gas chromatography–mass spectrometry. Additionally, urinary free cortisol (UFF) and cortisone (UFE) were quantified by RIA after extraction and chromatographic purification. Data on urinary steroids from 400 healthy controls served as reference values. Enzyme activities were assessed by established steroid metabolite ratios, e.g. 5α-reductase and 11beta-hydroxysteroid dehydrogenase Type 2 (11beta-HSD2) by 5α-tetrahydrocortisol/tetrahydrocortisol and UFE/UFF, respectively. Urinary markers of adrenarche, especially dehydroepiandrosterone and its direct metabolites were elevated in patients, as were urinary 6β-hydroxycortisol, UFE, and 11beta-HSD2 activity. However, overall cortisol secretion, as reflected by the sum of major urinary cortisol metabolites, was mostly normal and activity of 5α-reductase clearly reduced. Our study provides evidence for an exaggerated adrenarche in T1DM children, which may help to understand reported sequelae in female patients like hyperandrogenic symptoms. The findings also suggest a reduced cortisol inactivation via 5α-reductase that is not compensated by a fall in cortisol secretion. Whether the elevated urinary 6β-hydroxycortisol and cortisone excretion, observed in the patients, are also present in other forms of hypercortisolism and may thus serve as non-invasive clinical stress markers deserves further study.

Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep

Periconceptional undernutrition alters fetal growth, metabolism and endocrinology in late gestation. The underlying mechanisms remain uncertain, but fetal exposure to excess maternal glucocorticoids has been hypothesized. We investigated the effects of periconceptional undernutrition on maternal hypothalamic-pituitary-adrenal axis function and placental 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) activity. Ewes received maintenance feed (N, n= 20) or decreased feed from -60 to +30 days from mating to achieve 15% weight loss after an initial 2-day fast (UN, n= 21). Baseline plasma samples and arginine vasopressin (AVP)-corticotrophin-releasing hormone (CRH) challenges were performed on days -61, -57, -29, -1, +29, 33, and 49 from mating (day 0). Maternal adrenal and placental tissue was collected at 50 days. Baseline plasma levels of adrenocorticotrophic hormone (ACTH) and cortisol decreased in the UN group (P < 0.0001). ACTH response to AVP-CRH was greater in UN ewes during undernutrition (P= 0.03) returning to normal levels after refeeding. Cortisol response to AVP-CRH was greater in UN ewes after the initial 2-day fast, but thereafter decreased and was lower in UN ewes from mating until the end of the experiment (P= 0.007). ACTH receptor, StAR and p450c17 mRNA levels were down-regulated in adrenal tissue from UN ewes. Placental 11betaHSD2 activity was lower in UN than N ewes at 50 days (P= 0.014). Moderate periconceptional undernutrition results in decreased maternal plasma cortisol concentrations during undernutrition and after refeeding, and adrenal resistance to ACTH for at least 20 days after refeeding. Fetal exposure to excess maternal cortisol is unlikely during the period of undernutrition, but could occur later in gestation if maternal plasma cortisol levels return to normal while placental 11betaHSD2 activity remains low.

Enzymatic Properties of a Member (AKR1C20) of the Aldo-Keto Reductase Family

AKR1C20, a member of the aldo-keto reductase (AKR) superfamily, found by mouse genomic analysis, exhibits the highest sequence identity (89%) with mouse liver 17beta-hydroxysteroid dehydrogenase (HSD) type 5, but its function remains unknown. In this report, we have expressed the recombinant AKR1C20 from its cDNA, and examined its properties. The purified enzyme was a 36-kDa monomer, and showed both 17beta-HSD and 3alpha-HSD activities in the presence of NADP(H) as the coenzymes. While the Km values for testosterone and 5alpha-dihydrotestosterone were high (>0.2 mM), those for 3alpha-hydroxy- and 3-keto-steroids were low (0.3-5 microM), resulting in high catalytic efficiency for the substrates. Although no significant dehydrogenase activity towards non-steroidal alcohols was observed, the enzyme highly reduced alpha-dicarbonyl compounds such as 16-ketoestrone, 9,10-phenanthrenequinone, acenaphthenequinone, 1-phenylisatin and camphorquinone. The pH optima of the dehydrogenase and reductase activities were 10.5 and 6.5-7.5, respectively. The enzyme was inhibited by sulfobromophthalein, hexestrol, indomethacin and flufenamic acid. The properties of AKR1C20 are distinct from those of previously known mouse 17beta-HSD type 5 (AKR1C6), 3alpha-HSD (AKR1C14) and other members of the AKR1C subfamily. Thus, AKR1C20 is a novel 3alpha(17beta)-HSD, which may also function as a reductase for xenobiotic alpha-dicarbonyl compounds.

Evidence That the 11 β-Hydroxysteroid Dehydrogenase (11 β-HSD1) Is Regulated by Pentose Pathway Flux: STUDIES IN RAT ADIPOCYTES AND MICROSOMES

11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) catalyzes the interconversion of biologically inactive 11 keto derivatives (cortisone, 11-dehydrocorticosterone) to active glucocorticoids (cortisol, corticosterone) in fat, liver, and other tissues. It is located in the intraluminal compartment of the endoplasmic reticulum. Inasmuch as an oxo-reductase requires NADPH, we reasoned that 11 beta-HSD1 would be metabolically interconnected with the cytosolic pentose pathway because this pathway is the primary producer of reduced cellular pyridine nucleotides. To test this theory, 11 beta-HSD1 activity and pentose pathway were simultaneously measured in isolated intact rodent adipocytes. Established inhibitors of NAPDH production via the pentose pathway (dehydroandrostenedione or norepinephrine) inhibited 11 beta-HSD1 oxo-reductase while decreasing cellular NADPH content. Conversely these compounds slightly augmented the reverse, or dehydrogenase, reaction of 11 beta-HSD1. Importantly, using isolated intact microsomes, the inhibitors did not directly alter the tandem microsomal 11 beta-HSD1 and hexose-6-phosphate dehydrogenase enzyme unit. Metabolites of 11 beta-HSD1 (corticosterone or 11-dehydrocorticosterone) inhibited or increased pentose flux, respectively, demonstrating metabolic interconnectivity. Using isolated intact liver or fat microsomes, glucose-6 phosphate stimulated 11 beta-HSD1 oxo-reductase, and this effect was blocked by selective inhibitors of glucose-6-phosphate transport. In summary, we have demonstrated a metabolic interconnection between pentose pathway and 11 beta-HSD1 oxo-reductase activities that is dependent on cytosolic NADPH production. These observations link cytosolic carbohydrate flux with paracrine glucocorticoid formation. The clinical relevance of these findings may be germane to the regulation of paracrine glucocorticoid formation in disturbed nutritional states such as obesity.

Tibolone Metabolism in Human Liver Is Catalyzed by 3α/3β-Hydroxysteroid Dehydrogenase Activities of the Four Isoforms of the Aldo-Keto Reductase (AKR)1C Subfamily

Tibolone [[7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3alpha- and 3beta-hydroxymetabolites and a Delta4-isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3beta-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3alpha-hydroxytibolone. These observations may account for the appearance of 3beta-hydroxytibolone in target tissues and 3alpha-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3alpha- and 3beta-hydroxysteroid dehydrogenase (HSD) activity. 3beta-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5beta-cholanic acid-3alpha, 7alpha-diol. The cytosolic formation of 3alpha-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3beta-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3beta-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3beta- and 3alpha-hydroxytibolone was observed again in a 4:1 ratio. 3beta-HSD activity was inhibited by both 5beta-cholanic acid-3alpha,7alpha-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3alpha-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3beta- and 3alpha-Hydroxytibolone were rapidly metabolized into polar metabolites (>85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3alpha-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3alpha-HSD.

Comprehensive study of urinary cortisol metabolites in hyperthyroid and hypothyroid patients

To further analyse the significance and mutual relationship of thyroid function-linked alterations in cortisol metabolism that have been separately and variously reported. Twenty-four-hour urine samples from 21 patients with hyperthyroidism (Graves' disease), 16 patients with hypothyroidism (Hashimoto's thyroiditis), 21 healthy age- and sex-matched controls for hyperthyroidism, and 16 healthy age- and sex-matched controls for hypothyroidism were evaluated for 6beta-hydroxycortisol (6beta-OHF), tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-tetrahydrocortisol (allo-THF), urinary free cortisol (UFF), urinary free cortisone (UFE) and 17-hydroxycorticosteroid (17-OHCS). Urinary 17-OHCS, THE and allo-THF levels increased considerably in hyperthyroid patients compared to the controls, while UFF and THF showed no difference between the two groups. Urinary 6beta-OHF was significantly lower in the hyperthyroid patients than in the controls. Both the urinary allo-THF + THF/THE and the UFF/UFE ratios were significantly lower in the hyperthyroid patients than in the controls, whereas only the former was significantly higher in the hypothyroid patients than in the controls. The urinary allo-THF/THF ratio was significantly higher in the hyperthyroid patients and significantly lower in the hypothyroid patients than in the controls. In an analysis of pooled subjects including all groups (n = 64), free T4 levels correlated negatively (P < 0.0001) with the urinary allo-THF + THF/THE ratio but not with the UFF/UFE ratio. The serum levels of free T4 correlated positively (P < 0.0001) with the urinary allo-THF/THF ratio. The thyroid hormones seem to affect the total 11beta-HSD activity (allo-THF + THF/THE) more strongly than the renal 11beta-HSD2 activity (UFF/UFE). 5alpha-reductase activity (allo-THF/THF) is also enhanced in hyperthyroidism, while the reduction of urinary 6beta-OHF in hyperthyroidism might be a secondary effect of the altered activity of the total 11beta-HSD and 5alpha-reductase.

Hypoxia Blocks 11β-Hydroxysteroid Dehydrogenase Type 2 Induction in Human Trophoblast Cells During Differentiation by a Time-dependent Mechanism that Involves both Translation and Transcription

The present study was undertaken to determine (1) if hypoxia-induced down-regulation of placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by HSD11B2 gene) activity and protein in human trophoblast cells during in vitro differentiation was mediated at the level of HSD11B2 gene transcription; and (2) whether the reduced placental 11beta-HSD2 in pregnancies complicated with fetal growth restriction (FGR) was a consequence of intrinsic abnormalities in trophoblast cells. Trophoblast cells were isolated from uncomplicated pregnancies and those complicated with FGR at term, and cultured for up to 72 h under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under normoxia, 11beta-HSD2 activity and protein increased progressively over the 72 h culture period, which was accompanied by a corresponding rise in 11beta-HSD2 mRNA. As demonstrated previously, hypoxia blocked the increase in levels of both 11beta-HSD2 activity and protein within the first 24h. In contrast, although hypoxia also prevented the rise in 11beta-HSD2 mRNA, it did not do so until 48 h. This time-dependent effect of hypoxia on placental 11beta-HSD2 activity/protein and mRNA suggests a dual mechanism of action whereby hypoxia may induce a rapid down-regulation of 11beta-HSD2 protein synthesis, which occurs initially at the level of translation, and later extends to the level of transcription. Indeed, transient transfection studies demonstrated that hypoxia diminished HSD11B2 promoter activity. When trophoblast cells isolated from FGR placentas were cultured and allowed to differentiate under the same conditions, they not only exhibited a similar pattern of 11beta-HSD2 activity and mRNA expression but also responded to hypoxia similarly to those from normal placentas. This suggests that the reduced placental 11beta-HSD2 in FGR is not due to intrinsic abnormalities in trophoblast cells, but likely a result of extrinsic factors associated with FGR.

In vivo activity of 11β‐hydroxysteroid dehydrogenase type 1 and free fatty acid‐induced insulin resistance

Free fatty acids (FFAs) induce hepatic insulin resistance and enhance hepatic gluconeogenesis. Glucocorticoids (GCs) also stimulate hepatic gluconeogenesis. The aim of this study was to investigate whether the FFA-induced hepatic insulin resistance is mediated by increased activity of hepatic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), accompanied by elevated hepatic cortisol levels. Following a 10-h overnight fast, six healthy male volunteers were investigated. A euglycaemic hyperinsulinaemic clamp was performed during lipid or saline infusion. To assess hepatic 11beta-HSD1 activity, plasma cortisol levels were measured after oral administration of cortisone acetate during lipid or saline infusion. In addition, 11beta-HSD activities were determined in vivo by calculating the urinary ratios of GC metabolites. Lipid infusion increased FFAs (5.41 +/- 1.00 vs. 0.48 +/- 0.20 mmol/l; P < 0.005) and significantly increased insulin resistance [glucose infusion rate (GIR) 6.02 +/- 2.60 vs. 4.08 +/- 2.15 mg/kg/min; P < 0.005]. After lipid and saline infusions no changes in 11beta-HSD1 activity were found, neither by changes in cortisone acetate to cortisol conversion nor by differences in urinary free cortisol (UFF) or cortisone (UFE), 5beta-tetrahydrocortisol (THF), 5alpha-THF, cortisone (THE), UFF/UFE and (5alpha-THF + THF)/THE ratios. We found no change in hepatic and whole-body 11beta-HSD1 activity during acute FFA-induced insulin resistance. Further studies are necessary to clarify whether 11beta-HSD1 in muscle and adipose tissue is influenced by FFAs and whether 11beta-HSD1 is involved in other conditions of insulin resistance.

Association of Serum Lipid Concentrations, Insulin Resistance Index and Catch-Up Growth with Serum Cortisol/Cortisone Ratio by Liquid Chromatography Tandem Mass Spectrometry in Children Born Small for Gestational Age

Intrauterine growth restriction (IUGR) may influence adrenocortical function, lipid metabolism and glucose tolerance in later life. Both cortisol (F) synthesis and metabolism contribute to serum F concentrations. 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enzyme converts F to biologically inactive cortisone (E). Decreased 11beta-HSD2 activity has been suggested for a reason to IUGR and to its metabolic consequences. Our aim was to develop a specific liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for analysing serum F and E concentrations, to determine the F/E ratios, and to correlate them with serum lipid concentrations, insulin resistance index (HOMA-IR), and catch-up growth in children born small for gestational age (SGA). The mean serum F and E concentrations, and F/E ratios did not differ between the SGA and their control children at 12 y age. The SGA children in the highest F/E ratio quartile had poorer gain in height between 0-12 y, and higher serum total and LDL cholesterol levels than those with lower F/E ratios. In logistic regression analysis, high LDL cholesterol, high HOMA-IR, and early pubertal stage associated with high F/E ratio in the SGA children. In conclusion, our LC-MS/MS method enables a reliable measurement of both F and E concentrations from a single serum sample. High serum F/E ratio may be associated with IUGR, its metabolic consequences, and poor catch-up growth in a subset of SGA children.

Gonadotropin stimuli increase adrenodoxin immunoreactivity in the mitochondria of rat ovarian cells

Adrenodoxin is a component of the electron transfer system for mitochondrial cytochrome P450 in steroidogenic cells. To elucidate whether the steroidogenic state can modulate the amount and the distribution of adrenodoxin in the mitochondria, we determined immunohistochemically the distribution of adrenodoxin and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in ovaries of rats under hypo- and hyperfunctional states. In the ovaries of control rats, adrenodoxin was distributed in the cristae of round-shaped mitochondria of both interstitial and theca cells. In hypophysectomized rats, no adrenodoxin was found in the mitochondria of atrophied cells. Treatment of hypophysectomized rats with human chorionic gonadotropin (hCG) or Humegon restored immunostaining of adrenodoxin in the interstitial and theca cells, and rendered the vesicular cristae of C- or ring-shaped mitochondria surrounding the lipid droplets adrenodoxin-positive. The distribution of 3betaHSD activity detected by enzyme histochemistry in these ovaries matched well that of adrenodoxin immunostaining. Our results indicate that immunostaining intensity of adrenodoxin in the mitochondria reflects the steroidogenic state in rat ovarian cells.

Type 2 11β‐Hydroxysteroid Dehydrogenase Activity in Human Fallopian Tube and Correlation of Enzyme Levels with Endometrial Histopathology

The inter-conversion of hormonally active cortisol and inactive cortisone is catalyzed by 11beta-hydroxysteroid dehydrogenase (11beta-HSD). This conversion controls the level of active glucocorticoid concentration in tissues. As the fallopian tube plays a major role in the process of fertilization, we wanted to investigate whether 11beta-HSD is present in the human fallopian tube to control the glucocorticoid levels as in other tissues. Isthmic, ampullary and fimbrial portions of the fallopian tube are obtained from patients undergoing hysterectomy and salpingo-oopherectomy for symptomatic leomyomata uteri. 11beta-HSD activities were measured in the homogenates of the tube, cortisol as the steroid substrate. The enzyme activity was expressed as nanomolar cortisone formed per minute per gram of tissue (mean +/- S.D.). A significant level of 11beta-HSD activity in oxidation direction was found in all three parts of the tube. There is no significant difference in the distribution of the enzyme activity throughout the tube. When tubal 11beta-HSD activity was compared with endometrial histology, the enzyme activity is significantly lower in proliferative endometrium when compared with secretory endometrium (P = 0.002). The enzyme activity in inactive endometrium is significantly higher than the active endometrium (P = 0.05). The presence of 11beta-HSD throughout the fallopian tube and its correlation to endometrial histology is indicative of its probable role in controlling the glucocorticoid levels in the tissue, which in turn may influence the fertilization process.

High-Throughput Screening of 11β-Hydroxysteroid Dehydrogenase Type 1 in Scintillation Proximity Assay Format

11beta-Hydroxysteroid dehydrogenase type-1 (11beta-HSD1) is a potential target for the treatment of diabetes, obesity, and hyperlipidemia. This enzyme is mainly responsible for reactivating glucocorticoid hormone inside cells such as adipose cells and liver cells by converting the inactive cortisone to active cortisol. Enzyme assays for 11beta-HSD1 involve either a thin-layer chromatography or high-performance liquid chromatography step to separate cortisol from the substrate cortisone. This additional step is labor intensive and increases the assay time, which limits assay throughput. A homogenous scintillation proximity assay-based method has been recently developed that enables high-throughput screening of 11beta-HSD1 inhibitors. We have applied this novel 11beta-HSD1 assay to screening a large-size compound collection and identified several structural classes of lead compounds that selectively inhibit the activity of 11beta-HSD1.

Effect of Pro-inflammatory Cytokines on Expression and Activity of 11β-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Term Placental Trophoblast and Human Choriocarcinoma JEG-3 Cells

11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is thought to act as a placental barrier protecting the fetus from high levels of maternal cortisol. On the other hand, intrauterine infection is one of the main causes of preterm birth and adverse fetal outcome, and pro-inflammatory cytokines may contribute to these adverse effects. However, the effect of pro-inflammatory cytokines on 11beta-HSD2 is still not clear. Therefore, we have evaluated the effect of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on 11beta-HSD2 in cultured human placental trophoblast and in human choriocarcinoma JEG-3 cells. Placental trophoblast cells were isolated from human term placenta. Placental trophoblast cells and JEG-3 cells were treated with TNF-alpha (0.1-10 ng/mL) or IL-1beta (0.1-10 ng/mL). Real-time reverse transcription polymerase chain reaction and Western blot were used to study the regulation of 11beta-HSD2 expression. 11beta-HSD2 activity was determined by measuring the rate of cortisol to cortisone conversion in the culture medium using thin-layer chromatography (TLC). In placental trophoblast, TNF-alpha and IL-1beta down-regulated 11beta-HSD2 mRNA expression and activity (both P <.05). The protein level was decreased only with IL-1beta (P <.05). In JEG-3 cells, 11beta-HSD2 mRNA was decreased by TNF-alpha but up-regulated by IL-1beta, with no significant change in protein expression and activity. Our results suggest caution in interpreting data using JEG-3 cells. However, our studies with primary trophoblast suggest that TNF-alpha and IL-1beta may increase the amount of cortisol crossing to the placenta and fetal circulation by attenuating 11beta-HSD2 activity, potentially contributing to preterm labor and altered fetal outcome in uterine infection.

Comparison of crystal structures of human type 3 3α‐hydroxysteroid dehydrogenase reveals an “induced‐fit” mechanism and a conserved basic motif involved in the binding of androgen

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

Circulating cortisone levels are associated with biochemical markers of bone formation and lumbar spine BMD: the Hertfordshire Cohort Study

Cortisone is an endogenous corticosteroid that has negligible intrinsic glucocorticoid activity but can be converted to the active corticosteroid cortisol by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 11beta-HSD1 is expressed in osteoblasts and may play a role in determining susceptibility to glucocorticoid-induced osteoporosis. In intact osteoblasts enzyme activity, and thus cortisol generation, is dependent on substrate concentration with an almost linear increase in activity across the physiological range. We have therefore attempted to measure the impact of 11beta-HSD1 activity on bone in vivo by examining the association of circulating cortisone with bone markers, bone mineral density (BMD) and bone loss in a cohort of women and men. Baseline cross-sectional association study involving 135 women and 171 men aged 61-73 years from the Hertfordshire Cohort Study and a 4 year follow-up study examining changes in BMD. Serum cortisone, cortisol and osteocalcin, and urinary type I collagen cross-linked N-telopeptide (NTX) were measured at baseline. BMD at spine and hip was measured at baseline and 4 years later. In men serum cortisone levels were negatively correlated with serum osteocalcin (r = -0.20, P = 0.01); a similar relationship was seen in women (r = -0.16, P = 0.06). No correlation was seen between serum cortisone and urinary NTX (r = 0.03, P = 0.74 for women; r = -0.03, P = 0.72 for men). A negative correlation was observed between serum cortisone and spine BMD in women (r = -0.18, P = 0.04); a similar relationship was also seen in men (r =-0.14, P = 0.07). However, cortisone did not correlate with BMD at the femoral neck or total hip or changes in BMD at any site over time. In analyses adjusted for adiposity, osteoarthritis grade and a range of life-style variables, these relationships did not change substantially. All these relationships were independent of cortisol concentrations. The most plausible explanation for the association of circulating cortisone levels with osteocalcin is the presence of 11beta-HSD1 activity within osteoblasts. The measurement of serum cortisone may independently give insights into the action of glucocorticoids on bone.