Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.     

Type 2 11β-hydroxysteroid dehydrogenase in foetal and adult life

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyse the interconversion of active cortisol to inactive cortisone; 11β-HSD1 is a low affinity, NADP(H)-dependent dehydrogenase/oxo-reductase, and 11β-HSD2 a high affinity, NAD-dependent dehydrogenase. Because of the importance of 11β-HSD in regulating corticosteroid hormone action, we have analysed the distribution of the 11β-HSD isoforms in human adult and foetal tissues (including placenta), and, in addition have performed a series of substrate specificity studies on the novel, kidney 11β-HSD2 isoform. Using an RT-PCR approach, we failed to detect 11β-HSD1 mRNA in any human mid-gestational foetal tissues. In contrast 11β-HSD2 mRNA was present in foetal lung, adrenal, colon and kidney. In adult tissues 11β-HSD2 gene expression was confined to the mineralocorticoid target tissues, kidney and colon, whilst 11β-HSD1 was expressed predominantly in glucocorticoid target tissues, liver, lung, pituitary and cerebellum. In human kidney homogenates, 11-hydroxylated progesterone derivatives, glycyrrhetinic acid, corticosterone and the “end products” cortisone and 11-dehydrocorticosterone were potent inhibitors of the NAD-dependent conversion of cortisol to cortisone. Finally high levels of 11β-HSD2 mRNA and activity were observed in term placentae, which correlated positively with foetal weight. The tissue-specific distribution of the 11β-HSD isoforms is in keeping with their differential roles, 11β-HSD1 regulating glucocorticoid hormone action and 11β-HSD2 mineralocorticoid hormone action. The correlation of 11β-HSD2 activity in the placenta with foetal weight suggests, in addition, a crucial role for this enzyme in foetal development, possibly in mediating ontogeny of the foetal hypothalamo-pituitary-adrenal axis.     

3β-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5α-androstane-3β,17β-diol and dehydroepiandrosterone as substrates

3β-Hydroxysteroid dehydrogenase (3β-HSD)/Δ^5→4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3β-HSD/Δ^5→4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3β-HSD activity. For this purpose, we compared the efficiencies of a 3β-hydroxy-5-ene steroid (DHEA) and a 3β-hydroxy-5α-reduced steroid (5α-androstane-3β,17β-diol, 5α-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K_m) for 5α-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K_m of placental 3β-HSD for 5α-A-diol was in the range of 18 to 40 μmol/l (n = 3) vs 0.45 to 4 μmol/l for DHEA (n = 3); for the liver enzyme, 17 μmol/l for 5α-A-diol and 0.60 μmol/l for DHEA, and for the skin enzyme 14 and 0.18 μmol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5α-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K_ms and rates of product formation was obtained by use of purified placental 3β-HSD with DHEA, pregnenolone, and 3β-hydroxy-5α-androstan-17-one (epiandrosterone) as substrates: the K_m of 3β-HSD for DHEA was 2.8 μmol/l, for pregnenolone 1.9 μmol/l, and for epiandrosterone 25 μmol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein·min and, with epiandrosterone, 127 nmol/mg protein·min. With placental homogenate as the source of 3β-HSD, DHEA at a constant level of 5 μmol/l behaved as a competitive inhibitor when the radiolabeled substrate, [³H]5α-A-diol, was present in concentrations of 20 to 60 μmol/l, but a lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [³H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5α-A-diol (40 μmol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K_ms. Studies of inactivation of purified placental 3β-HSD/Δ^5→4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5α-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3β-hydroxy-5-ene steroids as substrates when compared with those obtained with 3β-hydroxy-5α-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3β-HSD activity by end products of metabolism of 3β-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3β-hydroxy-5-ene steroids—and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5α-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3β-HSD activity only (i.e. no Δ^5→4-isomerase).     

Purification of full-length recombinant human and rat type 1 11β-hydroxysteroid dehydrogenases with retained oxidoreductase activities

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a membrane-bound glycoprotein localized in the endoplasmic reticulum. This enzyme has a key role in regulating local tissue glucocorticoid concentration, acting in vivo predominantly as an oxidoreductase. Previous attempts to purify the native enzyme have yielded a protein without reductase activity. To facilitate detailed studies on its structure and regulation, we have developed a method to purify the full-length human and rat 11β-HSD1 with retention of their natural oxidoreductase activities. This procedure involved recombinant expression of these histidine-tagged enzymes in the yeast Pichia pastoris; large-scale culturing in a fermentor; and single-step purification by metal affinity chromatography. Both enzymes were 90–95% pure and exhibited dehydrogenase and reductase activities with K_M values in agreement with those reported in the literature.     

Effects of a high-salt diet on adipocyte glucocorticoid receptor and 11-β hydroxysteroid dehydrogenase 1 in salt-sensitive hypertensive rats

High-salt diets decrease insulin sensitivity in salt-sensitive hypertensive rats, and glucocorticoids promote adipocyte growth and may have pathophysiological roles in the metabolic syndrome. The aim of this study was to clarify the relationship between high-salt diet and the adipocyte glucocorticoid hormones in salt-sensitive hypertensive rats. Six-week-old Dahl salt-sensitive (DS) hypertensive rats and salt-resistant (DR) rats were fed a high-salt diet or a normal-salt diet for 4 weeks. Fasting blood glucose (FBG), serum adiponectin, plasma insulin, and corticosterone in plasma and in visceral adipose tissues, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activities in adipose tissues and glucose uptake in isolated muscle were measured. Animals underwent an oral glucose tolerance test (OGTT). The expression of mRNA for glucocorticoid receptor (GR), 11β-HSD1 and tumor necrosis factor-α (TNF-α) in adipose tissues were measured using a real-time PCR. A high-salt diet did not influence FBG; however, decreased 2-deoxy glucose uptake and plasma insulin during OGTT in DS rats. The high-salt diet increased significantly adipose tissue corticosterone concentration and 11β-HSD1 activities, gene expression for GR, 11β-HSD1 and TNF-α in adipose tissues in DS rats compared with DR rats (p < 0.05). The high-salt diet did not influence plasma corticosterone and serum adiponectin concentration in DS and DR rats. These results suggest that changes in GR and 11β-HSD1 in adipose tissue may contribute to insulin sensitivity in salt-sensitive hypertensive rats.     

Green Tea and One of Its Constituents,Epigallocatechine-3-gallate,Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.     

Placental 11β-hydroxysteroid dehydrogenase and the programming of hypertension

Excessive foetal exposure to glucocorticoids retards growth and “programmes” adult hypertension in rats. Placental 11β-hydroxysteroid dehydrogenase (11β-HSD), which catalyses the conversion of corticosterone and cortisol to inert 11 keto-products, normally protects the foetus from excess maternal glucocorticoids. In both rats and humans there is considerable natural variation in placental 11β-HSD, and enzyme activity correlates with birth weight. Moreover, inhibition of placental 11β-HSD in the rat reduces birth weight and produces hypertensive adult offspring, many months after prenatal treatment with enzyme inhibitors; these effects are dependent upon maternal adrenal products. These data suggest that placental 11β-HSD, by regulating foetal exposure to maternal glucocorticoids, crucially determines foeto-placental growth and the programming of hypertension. Maternal protein restriction during pregnancy also produces hypertensive offspring and selectively attenuates placental 11β-HSD activity. Thus, deficiency of the placental barrier to maternal glucocorticoids may represent a common pathway between the maternal environment and foeto-placental programming of later disease. These data may, at least in part, explain the human epidemiological observations linking early life events to the risk of subsequent hypertension. The recent characterization, purification and cDNA cloning of a distinct human placental 11β-HSD (type 2) will aid the further study of these intriguing findings.     

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.     

The mesenchymal α11β1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens

α11β1 constitutes the most recent addition to the integrin family and has been shown to display a binding preference for interstitial collagens found in mesenchymal tissues. We have previously observed that when α11β1 integrin is expressed in cells lacking endogenous collagen receptors, it can mediate PDGF-BB-dependent chemotaxis on collagen I in vitro. To determine in which cells PDGF and α11β1 might cooperate in regulating cell migration in vivo, we studied in detail the expression and distribution of α11 integrin chain in mouse embryos and tested the ability of PDGF isoforms to stimulate the α11β1-mediated cell migration of embryonic fibroblasts.Full-length mouse α11 cDNA was sequenced and antibodies were raised to deduced α11 integrin amino acid sequence. In the embryonic mouse head, α11 protein and RNA were localized to ectomesenchymally derived cells. In the periodontal ligament, α11β1 was expressed as the only detectable collagen-binding integrin, and α11β1 is thus a major receptor for cell migration and matrix organization in this cell population. In the remainder of the embryo, the α11 chain was expressed in a subset of mesenchymal cells including tendon/ligament fibroblasts, perichondrial cells, and intestinal villi fibroblasts. Most of the α11-expressing cells also expressed the α2 integrin chain, but no detectable overlap was found with the α1 integrin chain. In cells expressing multiple collagen receptors, these might function to promote a more stable cell adhesion and render the cells more resistant to chemotactic stimuli.Wild-type embryonic fibroblasts activated mainly the PDGF β receptor in response to PDGF-BB and migrated on collagens I, II, III, IV, V, and XI in response to PDGF-BB in vitro, whereas mutant fibroblasts that lacked α11β1 in their collagen receptor repertoire showed a stronger chemotactic response on collagens when stimulated with PDGF-BB. In the cellular context of embryonic fibroblasts, α11β1 is thus anti-migratory.We speculate that the PDGF BB-dependent cell migration of mesenchymal cells is tightly regulated by the collagen receptor repertoire, and disturbances of this repertoire might lead to unregulated cell migration that could affect normal embryonic development and tissue structure.     

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd.     

Tissue- and temporal-specific regulation of 11β-hydroxysteroid dehydrogenase type 1 by glucocorticoids in vivo

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. Short-term glucocorticoid excess upregulates 11β-HSD-1 in liver and hippocampus leading to suggestions that 11β-HSD-1 ameliorates the deleterious effects of glucocorticoid excess by its 11β-dehydrogenase activity. However the predominant activity of 11β-HSD-1 in vivo is 11β-reduction, thus generating active glucocorticoid. We have re-examined the time-course of glucocorticoid regulation of 11β-HSD-1 in the liver, hippocampus and kidney of adult male rats in vivo.Sham operation markedly reduced 11β-HSD-1 mRNA expression in all tissues, and reduced 11β-HSD bioactivity in liver and hippocampus when compared to untouched controls. Adrenalectomy reduced 11β-HSD-1 expression in all tissues in the short-term (7 days), followed by subsequent recovery of enzyme activity by 21 days in liver and hippocampus. Dexamethasone replacement of adrenalectomised rats attenuated the initial decrease in hepatic 11β-HSD-1 activity, but by 21 days dexamethasone reduced activity compared to control levels.Thus glucocorticoids regulate 11β-HSD-1 in a complex tissue- and temporal-specific manner. This pattern of regulation suggests glucocorticoids repress 11β-HSD-1 at least in the liver, a pattern of regulation more consistent with the evidence that 11β-HSD-1 is an 11β-reductase in vivo. Operational stress per se down-regulates 11β-HSD-1 which has implications for interpretation and design of in vivo studies of 11β-HSD-1.     

Differential estrogenic 17β-hydroxysteroid dehydrogenase activity and type 12 17β-hydroxysteroid dehydrogenase expression levels in preadipocytes and differentiated adipocytes

Estradiol (E2) is produced locally in adipose tissue and could play an important role in fat distribution and accumulation, especially in women. It is well recognized that aromatase is expressed in adipose tissue; however the identity of its estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD) partner is not identified. To gain a better knowledge about the enzyme responsible for the conversion of estrone into estradiol, we determined the activity and expression levels of known estrogenic 17β-HSDs, namely types 1, 7 and 12 17β-HSD in preadipocytes before and after differentiation into mature adipocytes using an adipogenic media. Estrogenic 17β-HSD activity was assessed using [¹⁴C]-labelled estrone, while mRNA expression levels of types 1, 7 and 12 17β-HSD were quantified using real-time PCR and protein expression levels of type 12 17β-HSD was determined using immunoblot analysis. The data indicate that there is a low conversion of E1 into E2 in preadipocytes; however this activity is increased 5-fold (p < 0.0001) in differentiated adipocytes. The increased estrogenic 17β-HSD activity is consistent with the increase in protein expression levels of 17β-HSD12.     

Endogenous inhibitors (GALFs) of 11beta-hydroxysteroid dehydrogenase isoforms 1 and 2: derivatives of adrenally produced corticosterone and cortisol

Two isoforms of 11β-HSD exist; 11β-HSD1 is bi-directional (the reductase usually being predominant) and 11β-HSD2 functions as a dehydrogenase, conferring kidney mineralocorticoid specificity. We have previously described endogenous substances in human urine, “glycyrrhetinic acid-like factors (GALFs)”, which like licorice, inhibit the bi-directional 11β-HSD1 enzyme as well as the dehydrogenase reaction of 11β-HSD2.

Many of the more potent GALFs are derived from two major families of adrenal steroids, corticosterone and cortisol. For example, 35-tetrahydro-corticosterone, its derivative, 35-tetrahydro-11β-hydroxy-progesterone (produced by 21-deoxygenation of corticosterone in intestinal flora); 35-tetrahydro-11β-hydroxy-testosterone (produced by side chain cleavage of cortisol); are potent inhibitors of 11β-HSD1 and 11β-HSD2-dehydrogenase, with IC₅₀'s in range 0.26–3.0 μM, whereas their 11-keto-35-tetrahydro-derivatives inhibit 11β-HSD1 reductase, with IC₅₀'s in range 0.7–0.8 μM (their 35β-derivatives being completely inactive).

Inhibitors of 11β-HSD2 increase local cortisol levels, permitting it to act as a mineralocorticoid in kidney. Inhibitors of 11β-HSD1 dehydrogenase/11β-HSD1 reductase serve to adjust the set point of local deactivation/reactivation of cortisol in vascular and other glucocorticoid target tissues, including adipose, vascular, adrenal tissue, and the eye. These adrenally derived 11-oxygenated C₂₁- and C₁₉-steroidal substances may serve as 11β-HSD1- or 11β-HSD2-GALFs. We conclude that adrenally derived products are likely regulators of local cortisol bioactivity in humans.     

Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Prevention of D-GalN/LPS-induced ALI by 18β-glycyrrhetinic acid through PXR-mediated inhibition of autophagy degradation

Acute liver injury (ALI) has multiple causes and results in liver dysfunction. Severe or persistent liver injury eventually leads to liver failure and even death. Pregnane X receptor (PXR)-null mice present more severe liver damage and lower rates of autophagy. 18β-glycyrrhetinic acid (GA) has been proposed as a promising hepatoprotective agent. We hypothesized that GA significantly alleivates D-GalN/LPS-induced ALI, which involved in PXR-mediated autophagy and lysosome biogenesis. We found that GA can significantly decrease hepatocyte apoptosis and increase the hepatic autophagy marker LC3-B. Ad-mCherry-GFP-LC3 tandem fluorescence, RNA-seq and real-time PCR indicated that GA may stabilize autophagosomes and lysosomes and inhibit autophagosome–lysosome fusion. Simultaneously, GA markedly activates PXR, even reversing the D-GalN/LPS-induced reduction of PXR and its downstream genes. In contrast, GA has a weak protective effect in pharmacological inhibition of PXR and PXR-null mice, which significantly affected apoptosis- and autophagy-related genes. PXR knockout interferes with the stability of autophagosomes and lysosomes, preventing GA reducing the expression of lysosomal genes such as Cst B and TPP1, and suppressing autophagy flow. Therefore, we believe that GA increases autophagy by inhibiting autophagosome–lysosome fusion and blocked autophagy flux via activation of PXR. In conclusion, our results show that GA activates PXR to regulate autophagy and lysosome biogenesis, represented by inhibiting autophagosome–lysosome fusion and stabilization of lysosome. These results identify a new mechanism by which GA-dependent PXR activation reduces D-GalN/LPS-induced acute liver injury.Subject terms: Metabolic disorders, Immunopathogenesis