Novel H6PDH mutations in two girls with premature adrenarche: 'apparent' and 'true' CRD can be differentiated by urinary steroid profiling.

CONTEXT: Inactivating mutations in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, encoded by H6PD) cause apparent cortisone reductase deficiency (ACRD). H6PDH generates cofactor NADPH for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by HSD11B1) oxo-reductase activity, converting cortisone to cortisol. Inactivating mutations in HSD11B1 cause true cortisone reductase deficiency (CRD). Both ACRD and CRD present with hypothalamic-pituitary-adrenal (HPA) axis activation and adrenal hyperandrogenism. OBJECTIVE: To describe the clinical, biochemical and molecular characteristics of two additional female children with ACRD and to illustrate the diagnostic value of urinary steroid profiling in identifying and differentiating a total of six ACRD and four CRD cases. DESIGN: Clinical, biochemical and genetic assessment of two female patients presenting during childhood. In addition, results of urinary steroid profiling in a total of ten ACRD/CRD patients were compared to identify distinguishing characteristics. RESULTS: Case 1 was compound heterozygous for R109AfsX3 and a novel P146L missense mutation in H6PD. Case 2 was compound heterozygous for novel nonsense mutations Q325X and Y446X in H6PD. Mutant expression studies confirmed loss of H6PDH activity in both cases. Urinary steroid metabolite profiling by gas chromatography/mass spectrometry suggested ACRD in both cases. In addition, we were able to establish a steroid metabolite signature differentiating ACRD and CRD, providing a basis for genetic diagnosis and future individualised management. CONCLUSIONS: Steroid profile analysis of a 24-h urine collection provides a diagnostic method for discriminating between ACRD and CRD. This will provide a useful tool in stratifying unresolved adrenal hyperandrogenism in children with premature adrenarche and adult females with polycystic ovary syndrome (PCOS).

Short- and long-term glucocorticoid treatment enhances insulin signalling in human subcutaneous adipose tissue.

BACKGROUND: Endogenous or exogenous glucocorticoid (GC) excess (Cushing's syndrome) is characterized by increased adiposity and insulin resistance. Although GCs cause global insulin resistance in vivo, we have previously shown that GCs are able to augment insulin action in human adipose tissue, contrasting with their action in skeletal muscle. Cushing's syndrome develops following chronic GC exposure and, in addition, is a state of hyperinsulinemia. OBJECTIVES: We have therefore compared the impact of short- (24 h) and long-term (7 days) GC administration on insulin signalling in differentiated human adipocytes in the presence of low or high concentrations of insulin. RESULTS: Both short- (24 h) and long-term (7 days) treatment of chub-s7 cells with dexamethasone (Dex) (0.5 µM) increased insulin-stimulated pTyr612IRS1 and pSer473akt/PKB, consistent with insulin sensitization. Chronic high-dose insulin treatment induced insulin resistance in chub-s7 cells. However, treatment with both high-dose insulin and Dex in combination still caused insulin sensitization. CONCLUSIONS: In this human subcutaneous adipocyte cell line, prolonged GC exposure, even in the presence of high insulin concentrations, is able to cause insulin sensitization. We suggest that this is an important mechanism driving adipogenesis and contributes to the obese phenotype of patients with Cushing's syndrome.

A novel selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis.

Glucocorticoid excess increases fat mass, preferentially within omental depots; yet circulating cortisol concentrations are normal in most patients with metabolic syndrome (MS). At a pre-receptor level, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) activates cortisol from cortisone locally within adipose tissue, and inhibition of 11beta-HSD1 in liver and adipose tissue has been proposed as a novel therapy to treat MS by reducing hepatic glucose output and adiposity. Using a transformed human subcutaneous preadipocyte cell line (Chub-S7) and human primary preadipocytes, we have defined the role of glucocorticoids and 11beta-HSD1 in regulating adipose tissue differentiation. Human cells were differentiated with 1.0 microM cortisol (F), or cortisone (E) with or without 100 nM of a highly selective 11beta-HSD1 inhibitor PF-877423. 11beta-HSD1 mRNA expression increased across adipocyte differentiation (P<0.001, n=4), which was paralleled by an increase in 11beta-HSD1 oxo-reductase activity (from nil on day 0 to 5.9+/-1.9 pmol/mg per h on day 16, P<0.01, n=7). Cortisone enhanced adipocyte differentiation; fatty acid-binding protein 4 expression increased 312-fold (P<0.001) and glycerol-3-phosphate dehydrogenase 47-fold (P<0.001) versus controls. This was abolished by co-incubation with PF-877423. In addition, cellular lipid content decreased significantly. These findings were confirmed in the primary cultures of human subcutaneous preadipocytes. The increase in 11beta-HSD1 mRNA expression and activity is essential for the induction of human adipogenesis. Blocking adipogenesis with a novel and specific 11beta-HSD1 inhibitor may represent a novel approach to treat obesity in patients with MS.

Expression profiling of 11beta-hydroxysteroid dehydrogenase type-1 and glucocorticoid-target genes in subcutaneous and omental human preadipocytes.

Obesity is associated with increased morbidity and mortality from cardiovascular disease, diabetes and cancer. Although obesity is a multi-factorial heterogeneous condition, fat accumulation in visceral depots is most highly associated with these risks. Pathological glucocorticoid excess (i.e. in Cushing's syndrome) is a recognised, reversible cause of visceral fat accumulation. The aim of this study was to identify depot-specific glucocorticoid-target genes in adipocyte precursor cells (preadipocytes) using Affymetrix microarray technique. Confluent preadipocytes from subcutaneous (SC) and omental (OM) adipose tissue collected from five female patients were treated for 24 h with 100 nM cortisol (F), RNA was pooled and hybridised to the Affymetrix U133 microarray set. We identified 72 upregulated and 30 downregulated genes by F in SC cells. In OM preadipocytes, 56 genes were increased and 19 were decreased. Among the most interesting were transcription factors, markers of adipocyte differentiation and glucose metabolism, cell adhesion and growth arrest protein factors involved in G-coupled and Wnt signalling. The Affymetrix data have been confirmed by quantitative real-time PCR for ten specific genes, including HSD11B1, GR, C/EBPalpha, C/EBPbeta, IL-6, FABP4, APOD, IRS2, AGTR1 and GHR. One of the most upregulated genes in OM but not in SC cells was HSD11B1. The GR was similarly expressed and not regulated by glucocorticoids in SC and OM human preadipocytes. C/EBPalpha was expressed in SC preadipocytes and upregulated by F, but was below the detection level in OM cells. C/EBPbeta was highly expressed both in SC and in OM preadipocytes, but was not regulated by F. Our results provide insight into the genes involved in the regulation of adipocyte differentiation by cortisol, highlighting the depot specifically in human adipose tissue.

Expression of 11beta-hydroxysteroid dehydrogenase (11betaHSD) proteins in luteinizing human granulosa-lutein cells.

In a range of tIssues, cortisol is inter-converted with cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To date, two isoforms of 11betaHSD have been cloned. Previous studies have shown that human granulosa cells express type 2 11betaHSD mRNA during the follicular phase of the ovarian cycle, switching to type 1 11betaHSD mRNA expression as luteinization occurs. However, it is not known whether protein expression, and 11betaHSD enzyme activities reflect this reported pattern of mRNA expression. Hence, the aims of the current study were to investigate the expression and activities of 11betaHSD proteins in luteinizing human granulosa-lutein (hGL) cells. Luteinizing hGL cells were cultured for up to 3 days with enzyme activities (11beta-dehydrogenase (11betaDH) and 11-ketosteroid reductase (11 KSR)) and protein expression (type 1 and type 2 11betaHSD) assessed on each day of culture. In Western blots, an immunopurified type 1 11betaHSD antibody recognized a band of 38 kDa in hGL cells and in human embryonic kidney (HEK) cells stably transfected with human type 1 11betaHSD. The type 2 11betaHSD antibody recognized a band of 48 kDa in HEK cells transfected with human type 2 11betaHSD cDNA but the type 2 protein was not expressed in hGL cells throughout the 3 days of culture. While the expression of type 1 11betaHSD protein increased progressively by 2.7-fold over 3 days as hGL cells luteinized, both 11betaDH and reductase activities declined (by 52.9% and 34.2%; P<0.05) over this same period. Changes in enzyme expression and activity were unaffected by the suppression of ovarian steroid synthesis.

Expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue is not increased in human obesity.

Central obesity is associated with increased morbidity and mortality. Preadipocyte proliferation and differentiation contribute to increases in adipose tissue mass, yet the mechanisms that underlie these processes remain unclear. Patients with glucocorticoid excess develop a reversible form of central obesity, but circulating cortisol levels in idiopathic obesity are invariably normal. We have hypothesized that the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), by converting inactive cortisone to active cortisol in adipose tissue, might be an important autocrine regulator of fat mass. Paired omental and sc fat biopsies were obtained from 32 women (median age, 43 yr; range, 28-65; median body mass index, 27.5 kg/m(2); range, 19.7-39.2) undergoing elective abdominal surgery. 11beta-HSD1 activity and mRNA levels were assessed in whole tissue and in isolated preadipocytes and adipocytes using specific enzyme assays and real-time PCR. Preadipocyte proliferation was measured using tritiated thymidine incorporation. Whole adipose tissue 11beta-HSD1 mRNA levels did not differ between omental and sc samples (P = 0.73). In addition, mRNA levels did not correlate with body mass index (omental: r = 0.1; P = 0.6; sc: r = 0.15; P = 0.4). In keeping with earlier studies, 11beta-HSD1 mRNA levels were higher in omental compared with sc preadipocytes. However, in cultured omental preadipocytes, 11beta-HSD1 activity inversely correlated with body mass index (r = -0.47; P = 0.03). In omental preadipocytes, both cortisol and cortisone decreased proliferation (P < 0.05). Inhibition of 11beta-HSD1 with glycyrrhetinic acid partially reversed the cortisone-induced decrease in preadipocyte proliferation (P < 0.05). Enhanced preadipocyte proliferation within omental adipose tissue as a consequence of decreased 11beta-HSD1 mRNA levels and activity may contribute to increases in visceral adipose tissue mass in obese patients.

Modulation of 11beta-hydroxysteroid dehydrogenase isozymes by proinflammatory cytokines in osteoblasts: an autocrine switch from glucocorticoid inactivation to activation.

Tissue damage by proinflammatory cytokines is attenuated at both systemic and cellular levels by counter anti-inflammatory factors such as corticosteroids. Target cell responses to corticosteroids are dependent on several factors including prereceptor regulation via local steroidogenic enzymes. In particular, two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11beta-HSD1 by converting E to F and 11beta-HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11beta-HSD isozymes have been shown to be expressed in osteoblasts (OBs). Using the MG-63 human osteosarcoma cell-line and primary cultures of human OBs, we have studied the regulation of osteoblastic 11beta-HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG-63 cells, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) potently inhibited 11beta-HSD2 activity (cortisol-cortisone conversion) and messenger RNA (mRNA) levels in a dose-dependent manner while stimulating reciprocal expression of 11beta-HSD1 mRNA and activity (cortisone-cortisol conversion). A similar rise in 11beta-HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF-alpha. Pretreatment of MG-63 cells with 0.1 ng/ml IL-1beta resulted in increased cellular sensitivity to physiological glucocorticoids as shown by induction of serum and glucocorticoid-inducible kinase (SGK; relative increase with 50 nM F but no IL-1beta pretreatment 1.12 +/- 0.34; with pretreatment 2.63 +/- 0.50; p < 0.01). These results highlight a novel mechanism within bone cells whereby inflammatory cytokines cause an autocrine switch in intracellular corticosteroid metabolism by disabling glucocorticoid inactivation (11beta-HSD2) while inducing glucocorticoid activation (11beta-HSD1). Therefore, it can be postulated that some of the effects of proinflammatory cytokines within bone (e.g., periarticular erosions in inflammatory arthritis) are mediated by this mechanism.

Expression of 11 beta-hydroxysteroid dehydrogenase isoenzymes in the human pituitary: induction of the type 2 enzyme in corticotropinomas and other pituitary tumors.

One of the defining biochemical features of Cushing's disease is a relative insensitivity to glucocorticoid (GC) feedback, but an analysis of the GC receptor has failed to detect any major abnormalities. However, two isoenzymes of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD), either by converting cortisone (E) to cortisol (F) (type 1) or conversely by converting F to E (type 2), play an important prereceptor role in regulating corticosteroid hormone action at several sites. 11 beta HSD1 and -2 expression within the anterior pituitary gland itself may modulate GC feedback at an autocrine level, and we have speculated that this may be deranged in Cushing's disease. Detection of 11 beta HSD type 1 and 2 immunoreactive protein was performed using fluorescence immunohistochemistry. Double immunofluorescent studies were undertaken on normal pituitary to define the cellular localization of 11 beta HSD isoenzymes using antisera against GH, ACTH, LH, FSH, PRL, and S100, a nonhormonal marker of folliculo-stellate cells. In normal pituitary, positive staining for 11 beta HSD1-immunoreactive protein was observed in GH- and PRL-secreting cells and in folliculo-stellate cells; gonadotrophs, thyrotrophs, and ACTH-positive cells were negative. 11 beta HSD2 immunoreactivity was absent in all cell types. RT-PCR detected 11 beta HSD1 messenger ribonucleic acid (mRNA) expression in the normal pituitary; 11 beta HSD2 mRNA expression was also seen in most normal tissue. By contrast, in ACTH-secreting adenomas 11 beta HSD2 immunostaining was strongly positive in every case of corticotroph adenoma. 11 beta HSD1 immunoreactivity was also observed occasionally, but to a much lesser extent. In other pituitary tumors, both functional and nonfunctional, 11 beta HSD expression was variable in terms of isoenzyme mRNA and intensity of protein staining. The expression of 11 beta HSD1 (which generates F from E) in somatotrophs and lactotrophs suggests an autocrine role for this isoenzyme in the glucocorticoid regulation of pituitary GH and PRL secretion. 11 beta HSD2 expression is markedly induced in ACTH-secreting pituitary tumors and, by converting F to E, may explain the resetting of glucocorticoid feedback control in Cushing's disease.

Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines.

Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.

Expression of 11 beta-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies.

Interconversion of active and inactive glucocorticoids, e.g. cortisol (F) and cortisone (E) is catalysed by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) which exists as two isoforms. We have used human placental bed biopsies and an in-vitro cytotrophoblast cell culture system to examine the expression and activity of the 11 beta-HSD isoforms along with that of the glucocorticoid and mineralocorticoid receptors (GR and MR). Immunohistochemistry localized 11 beta-HSD1 to decidualized stromal cells and 11 beta-HSD2 to villous cytotrophoblast, syncytiotrophoblasts and trophoblast cells invading the placental bed and maternal vasculature. In primary cultures of human cytotrophoblast, 11 beta-HSD2, GR and MR mRNA were expressed. Low levels of 11 beta-HSD1 mRNA were noted in these cultured cells, but could be explained on the basis of contaminating, vimentin-positive decidual stromal cells (< or =5%). Enzyme activity studies confirmed the presence of a high-affinity, NAD-dependent dehydrogenase activity (K(m) 137 nmol/l and V(max) 128 pmol E/h/mg protein), indicative of the 11 beta-HSD2 isoform. No reductase activity was observed. The presence of functional MR and GR was determined using Scatchard analyses of dexamethasone and aldosterone binding (MR K(d) 1.4 nmol/l B(max) 3.0; GR K(d) 6.6 nmol/l B(max) 16.2 fmol/ng protein). The expression of 11 beta-HSD1 in maternal decidua and 11 beta-HSD2 in adjacent trophoblast suggests an important role for glucocorticoids in determining trophoblast invasion. The presence of the MR within trophoblast indicates that some of the effects of cortisol could be MR- rather than GR-mediated.

The role of 11 beta-hydroxysteroid dehydrogenase in central obesity and osteoporosis.

Both central obesity and osteoporosis are common findings in states of glucocorticoid excess. In many tissues, including adipose tissue, hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyses the inter-conversion of active glucocorticoid, cortisol (F) and inactive cortisone (E) and regulates exposure to the glucocorticoid receptor. As such, factors which regulate 11beta-HSD1 are likely to have an important role in adipose tissue and bone physiology. Using primary cultures of human adipose stromal cells we have investigated the effect of various factors present within the adipocyte microenvironment for their effects on 11beta-HSD1 expression. IGF-1 caused a dose dependant inhibition of 11beta-HSD1 activity in both subcutaneous and omental stromal cells. Additionally, TNFalpha treatment increased 11beta-HSD1 reductase activity and mRNA expression. In adult human bone, 11beta-HSD1, but not 11beta-HSD2, expression was demonstrated using enzyme activity studies, RT-PCR and immunohistochemistry. In contrast to liver and adipose tissues, where reductase activity predominates, both reductase and dehydrogenase activities of 11beta-HSD1 were evident in bone chips and primary cultures of human osteoblasts. The action of growth factors and cytokines on glucocorticoid sensitive tissues such as adipose tissue and bone may be mediated by modulation of local glucocorticoid metabolism at a pre-receptor level.

Differentiation of adipose stromal cells: the roles of glucocorticoids and 11beta-hydroxysteroid dehydrogenase.

Glucocorticoids play an important role in determining adipose tissue distribution and function, with glucocorticoid excess states such as Cushing's syndrome resulting in central obesity. We have investigated the functional significance of local generation of cortisol within adipose tissue from inactive cortisone through the activity of the NADP(H)-dependent enzyme, 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). In primary cultures of paired omental (om) and sc human adipose stromal cells (ASC; n = 34), 11betaHSD1 oxo-reductase activity was significantly higher in om ASC (median, 40.2 pmol/mg protein x h; 95% confidence interval, 1.8-105) compared with sc ASC (median, 11.4; 95% confidence interval, 0-48.1; P<0.001) despite similar endogenous NADPH/NADP concentrations. Both cortisol and insulin increased the differentiation of ASC to adipocytes (as assessed by glycerol-3-phosphate dehydrogenase expression), but only cortisol increased 11betaHSD1 activity and messenger RNA levels in a dose-dependent fashion. Cortisone (500 nM) was as effective as 500 nM cortisol in inducing ASC differentiation, but this stimulatory effect was inhibited by coincubation with the 11betaHSD1 inhibitor, glycyrrhetinic acid. The higher local conversion of cortisone to active cortisol through expression of 11betaHSD1 in om compared with sc ASC may explain the specific action of glucocorticoids on different adipose tissue depots. 11betaHSD1 expression in om ASC is regulated at a transcriptional level and is increased by glucocorticoids, but is not entirely dependent upon ASC differentiation. Inhibition of 11betaHSD1 within om ASC inhibits cortisone-induced ASC differentiation. These findings indicate that local metabolism of glucocorticoid may control differentiation of adipose tissue in a site-specific fashion. Specific inhibitors of 11betaHSD1 may offer a novel approach for the treatment of patients with central obesity.

Characterization of 11beta-hydroxysteroid dehydrogenase activity and corticosteroid receptor expression in human osteosarcoma cell lines.

Studies in vitro and in vivo have shown that corticosteroids play an important role in bone physiology and pathophysiology. It is now established that corticosteroid hormone action is regulated, in part, at the pre-receptor level through the expression of isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which are responsible for the interconversion of hormonally active cortisol to cortisone. In this report we demonstrate 11beta-HSD activity in human osteoblast (OB) cells. Osteosarcoma-derived OB cell lines TE-85, MG-63 and SaOS-2 and fibrosarcoma Hs913T cells express the type 2 isoform of 11beta-HSD, as determined by reverse transcription polymerase chain reaction (RT-PCR) and specific enzyme assays. Enzyme activity was shown to be strictly NAD dependent with a Km of approximately 71 nM; 11beta-HSD type 1 mRNA expression and enzyme activity were not detected. All four cell lines expressed mRNA for the glucocorticoid receptor (GR) and mineralocorticoid receptor, but specific binding was only detectable with radiolabelled dexamethasone (Kd=10 nM) and not aldosterone. MG-63 cells had two to three times more GR than the other OB cells, which correlated with the higher levels of 11beta-HSD 2 activity in these cells. In contrast to the osteosarcoma cell studies, RT-PCR analysis of primary cultures of human OB cells revealed the presence of mRNA for 11beta-HSD 1 as well as 11beta-HSD 2. However, enzyme activity in these cells remained predominantly oxidative, i.e. inactivation of cortisol to cortisone (147 pmol/h per mg protein at 500 nM cortisol) was greater than cortisone to cortisol (10.3 pmol/h per mg protein at 250 nM cortisone). Data from normal human OB and osteosarcoma cells demonstrate the presence of an endogenous mechanism for inactivation of glucocorticoids in OB cells. We postulate that expression of the type 1 and type 2 isoforms of 11beta-HSD in human bone plays an important role in normal bone homeostasis, and may be implicated in the pathogenesis of steroid-induced osteoporosis.

Characterization of aromatase and 17 beta-hydroxysteroid dehydrogenase expression in rat osteoblastic cells.

Postmenopausal loss of 17 beta-estradiol (E2) in women is associated with decreased bone mineral density and increased susceptibility to osteoporotic bone fracture. These changes in bone status are assumed to be due to circulating levels of the hormone; therapeutic replacement of E2 can alleviate the bone disease. However, recent reports have shown that human osteoblastic (OB) cells are able to synthesize estrogens locally, via expression of the enzyme aromatase. In this study, we have characterized the expression and activity of aromatase and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in rat OB cell lines. Aromatase activity in ROS 17/2.8, ROS 25/1, and UMR 106 cells was similar to that shown in human OB cells, with the highest levels of activity observed in the more differentiated ROS 17/2.8 cells (Vmax = 45 pmol/h/mg of protein). The rat OB cells also showed 17 beta-HSD activity, with the predominant metabolism in all three cell lines being estrone (E1) to E2. As with aromatase, the highest activity was observed in ROS 17/2.8 cells (Vmax = 800 pmol/h/mg of protein). Northern analyses indicated the variable presence of transcripts corresponding to the type 1, 2, 3, and 4 isoforms of 17 beta-HSD. Further analysis of androstenedione metabolism indicated that the net effect of aromatase and 17 beta-HSD activity varied with cell type and culture treatment. All three OB cell lines were able to synthesize E1, E2, and testosterone from androstenedione, although activity varied between OB cell types. Regulatory effects were observed with 1,25-dihydroxyvitamin D3 (positive) and dexamethasone (negative). These data suggest that local synthesis of sex hormones is an important function of OB cells and may play a key role in the modulation of bone turnover independent of circulating hormone concentrations.

Immunohistochemical localization of type 1 11beta-hydroxysteroid dehydrogenase in human tissues.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11betaHSD) catalyze the interconversion of hormonally active cortisol to inactive cortisone. Activity and messenger ribonucleic acid studies indicate that type 1 11betaHSD (11betaHSD1) is expressed in glucocorticoid target tissues such as liver, gonad, and cerebellum, where it regulates the exposure of cortisol to glucocorticoid receptors. To further understand the role of 11betaHSD1 in human tissues, we have studied the localization of this isozyme using an antibody raised in sheep against amino acids 19-33 of human 11betaHSD1. Western blot analyses indicated that the immunopurified antibody recognized a band of approximately 34 kDa in human liver and decidua. Immunoperoxidase studies on liver, adrenal, ovary, decidua, and adipose tissue indicated positive cytoplasmic staining for 11betaHSD1. 11BetaHSD1 immunoreactivity was observed more intensely around the hepatic central vein, with no staining around the portal vein, hepatic artery, or bile ducts. No staining for 11betaHSD1 was observed in the adrenal medulla, but 11betaHSD1-immunoreactive protein was observed in all three zones of the adrenal cortex, with the most intense staining in the zona reticularis > zona glomerulosa > zona fasciculata. In the human ovary, immunoreactivity was observed in the developing oocyte and the luteinized granulosa cells of the corpus luteum. No staining was observed in granulosa cells, thecal cells, or ovarian stroma, which contrasted with the marked expression of 11betaHSD2 in the granulosa cell layer. Sections of human decidua showed high expression of 11betaHSD1 in decidual cells. In omental adipose tissue, 11betaHSD1 immunoreactivity was observed in both stromal and adipocyte cells. Immunohistochemical localization of 11betaHSD1 in human liver, adrenal, ovary, decidua, and adipose tissue using this novel antiserum provides us with a tool to investigate the role of this isozyme in modulating glucocorticoid hormone action within these tissues.

Isoforms of 11beta-hydroxysteroid dehydrogenase in human granulosa-lutein cells.

To date, two isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been characterized: a low affinity, NADP+-dependent isoform (11betaHSD1) and a high affinity, NAD+-dependent isoform which metabolizes dexamethasone and is inhibited by cortisone (11betaHSD2). Having previously reported a relationship between ovarian 11betaHSD activities and conception in women undergoing in vitro fertilization (IVF-ET), the objective of the present study was to identify which isoforms of 11betaHSD metabolize glucocorticoids in cultures of human granulosa-lutein cells. In both intact cells and cell homogenates, two distinct 11betaHSD activities were identified with differing affinities for cortisol (Km = 490 nM and 2.6 microM). Even at low concentrations, cortisol oxidation was preferentially supported by NADP+ and was independent of NAD+. Although inhibited by the hemisuccinate ester of glycyrrhetinic acid, carbenoxolone, the predominant 11betaHSD activity in intact cells was resistant to end-product inhibition. Intact cells were also able to reduce [3H]cortisone (Km = 190 nM) but did not metabolize [3H]dexamethasone. 11BetaHSD1 mRNA was expressed in 23 of 28 cell cultures whereas 11betaHSD2 mRNA was not expressed in any of the 22 independent cultures studied by reverse transcriptase-polymerase chain reaction (RT-PCR). We conclude that human granulosa-lutein cells express both type 11betaHSD and a novel isoform of this enzyme. While the low affinity 11beta-dehydrogenase and 11-ketosteroid reductase activities exhibit properties consistent with 11betaHSD1, the high affinity 11beta-dehydrogenase differs from 11betaHSD2 in that it is NADP+-dependent, does not metabolize dexamethasone and is resistant to end-product inhibition.

Does central obesity reflect "Cushing's disease of the omentum"?

BACKGROUND: Central obesity results in a cluster of metabolic abnormalities contributing to premature death. Glucocorticoids regulate adipose-tissue differentiation, function, and distribution, and in excess, cause central obesity. Glucocorticoid hormone action is, in part, controlled by two isoforms of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) which interconverts hormonally active cortisol to inactive cortisone. We studied cortisol metabolism within different adipose tissue depots. METHODS: We analysed expression and activity of the two isoforms (1 and 2) of 11 beta-HSD in cultured omental and subcutaneous adipose stromal cells from 16 patients undergoing elective abdominal surgery. FINDINGS: Only the type 1 isoform (11 beta-HSD1) was expressed in adipose stromal cells. The predominant activity was oxo-reductase (conversion of cortisone to cortisol greater than cortisol to cortisone) and was higher in omental than subcutaneous fat (cortisone to cortisol, median 57.6 pmol mg-1 h-1 [95% CI 25.8-112.9] vs 0 pmol mg-1 h-1 [0-0.6], p < 0.001). 11 beta-HSD1 oxo-reductase activity was further increased (127.5 pmol mg-1 h-1 [82.1-209], p < 0.05) when omental adipose stromal cells were treated with cortisol and insulin. INTERPRETATION: Adipose stromal cells from omental fat, but not subcutaneous fat, can generate active cortisol from inactive cortisone through the expression of 11 beta-HSD1. The expression of this enzyme is increased further after exposure to cortisol and insulin. In vivo, such a mechanism would ensure a constant exposure of glucocorticoid specifically to omental adipose tissue, suggesting that central obesity may reflect "Cushing's disease of the omentum".

Human 11 beta-hydroxysteroid dehydrogenase: studies on the stably transfected isoforms and localization of the type 2 isozyme within renal tissue.

The type 1 and type 2 isoforms of human 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) play a crucial role, respectively, in modulating glucocorticoid and mineralocorticoid hormone action. Deficiency of the 11 beta-HSD2 isoform, as described in the syndrome of apparent mineralocorticoid excess and following liquorice (glycyrrhetinic acid) or carbenoxolone ingestion, results in hypertension in which cortisol acts as a potent mineralocorticoid. Several studies have addressed the effects of progesterone, glycyrrhetinic acid, and their derivatives on 11 beta-HSD activity, but these were largely undertaken before the characterization of the 11 beta-HSD isoforms. The aim of this study was to evaluate the localization of 11 beta-HSD2 in human kidney and to study the effects of progesterone, glycyrrhetinic acid, and their related compounds on stable transfectants of the human 11 beta-HSD isoforms. Using an in-house sheep antibody against human 11 beta-HSD2, immunoperoxidase studies localized 11 beta-HSD2 to renal cortical and medullary collecting ducts. Glomeruli, vascular structures, loops of Henle, and proximal tubules were all negative. Confocal laser microscopy studies indicated both a cytoplasmic and nuclear localization for the enzyme within renal collecting ducts. The nuclear staining, which was intranuclear and was not associated with the nuclear membrane, accounted for 40% of the total cellular 11 beta-HSD2 immunoreactivity. Kinetic analysis of 11 beta-HSD activity in fetal kidney 293 cells stably transfected with h11 beta-HSD1/pcDNA3 or 11 beta-HSD2/pCR3, indicated, respectively, low-affinity dehydrogenase/oxoreductase activity (Km for F, 1.8 microM; Km for E, 270 nM) and high-affinity dehydrogenase activity (Km for F, 190 nM). The reductase activity of 11 beta-HSD1 was inhibited by 11 alpha-hydroxyprogesterone > carbenoxolone = glycyrrhetinic acid = progesterone > 11 beta-hydroxyprogesterone. The dehydrogenase activity of 11 beta-HSD2 was inhibited 11 alpha-hydroxyprogesterone = 11 beta-hydroxyprogesterone > glycyrrhetinic acid > carbenoxolone = progesterone. 11 beta-HSD2, expressed in the renal collecting duct, serves to protect the mineralocorticoid receptor (MR) in an autocrine fashion. The demonstration of a nuclear localization for what was thought to be principally a microsomal enzyme suggests that interaction between the MR and its ligand (either aldosterone or cortisol) may be a nuclear rather than a cytoplasmic event. The inhibitory effects of progesterone, glycyrrhetinic acid, and related compounds on 11 beta-HSD1 and 2 were similar, and it remains to be seen what implication these findings have for 11 beta-HSD1 action in tissues such as the liver and gonad and renal 11 beta-HSD2 activity in relation to sodium homeostasis and blood pressure control.

Glucose stimulates the activity of the guanine nucleotide-exchange factor eIF-2B in isolated rat islets of Langerhans.

Over short time periods glucose controls insulin biosynthesis predominantly through effects on preexisting mRNA. However, the mechanisms underlying the translational control of insulin synthesis are unknown. The present study was carried out to determine the effect of glucose on the activity and/or phosphorylation status of eukaryotic initiation and elongation factors in islets. Glucose was found to increase the activity of the guanine nucleotide-exchange factor eIF-2B over a rapid time course (within 15 min) and over the same range of glucose concentrations as those that stimulate insulin synthesis (3-20 mM). A nonmetabolizable analogue of glucose (mannoheptulose), which does not stimulate insulin synthesis, failed to activate eIF-2B. The best characterized mechanism for modulating eIF-2B activity involves changes in the phosphorylation of the alpha-subunit of its substrate eIF-2. However, in islets, no change in eIF-2 alpha phosphorylation was seen under conditions where eIF-2B activity was increased, implying that glucose regulates eIF-2B via an alternative pathway. Glucose also did not affect the phosphorylation states of three other regulatory translation factors. These are the cap-binding factor eIF-4E, 4E-binding protein-1, and elongation factor eEF-2, which do not therefore seem likely to be involved modulating the translation of the preproinsulin mRNA under these conditions.