Reduction Of Cortisone Research Articles

Age-related increase in the expression of 11β-hydroxysteroid dehydrogenase type 1 in the hippocampus of male rhesus macaques.

The hippocampus is especially susceptible to age-associated neuronal pathologies, and there is concern that the age-associated rise in cortisol secretion from the adrenal gland may contribute to their etiology. Furthermore, because 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) catalyzes the reduction of cortisone to the active hormone cortisol, it is plausible that an increase in the expression of this enzyme enhances the deleterious impact of cortisol in the hippocampus and contributes to the neuronal pathologies that underlie cognitive decline in the elderly. Rhesus macaques were used as a translational animal model of human aging, to examine age-related changes in gene and protein expressions of (HSD11B1/HSD11B1) in the hippocampus, a region of the brain that plays a crucial role in learning and memory. Older animals showed significantly (p < 0.01) higher base-line cortisol levels in the circulation. In addition, they showed significantly (p < 0.05) higher hippocampal expression of HSD11B1 but not NR3C1 and NR3C2 (i.e., two receptor-encoding genes through which cortisol exerts its physiological actions). A similar age-related significant (p < 0.05) increase in the expression of the HSD11B1 was revealed at the protein level by western blot analysis. The data suggest that an age-related increase in the expression of hippocampal HSD11B1 is likely to raise cortisol concentrations in this cognitive brain area, and thereby contribute to the etiology of neuropathologies that ultimately lead to neuronal loss and dementia. Targeting this enzyme pharmacologically may help to reduce the negative impact of elevated cortisol concentrations within glucocorticoid-sensitive brain areas and thereby afford neuronal protection.

Δ1-Dehydrogenation and C20 Reduction of Cortisone and Hydrocortisone Catalyzed by Rhodococcus Strains.

Prednisone and prednisolone are steroids widely used as anti-inflammatory drugs. Development of the pharmaceutical industry is currently aimed at introducing biotechnological processes and replacing multiple-stage chemical syntheses. In this work we evaluated the ability of bacteria belonging to the Rhodococcus genus to biotransform substrates, such as cortisone and hydrocortisone, to obtain prednisone and prednisolone, respectively. These products are of great interest from a pharmaceutical point of view as they have higher anti-inflammatory activity than the starting substrates. After an initial lab-scale screening of 13 Rhodococcus strains, to select the highest producers of prednisone and prednisolone, we reported the 200 ml-batch scale-up to test the process efficiency and productivity of the most promising Rhodococcus strains. R. ruber, R. globerulus and R. coprophilus gave the Δ1-dehydrogenation products of cortisone and hydrocortisone (prednisone and prednisolone) in variable amounts. In these biotransformations, the formation of products with the reduced carbonyl group in position C20 of the lateral chain of the steroid nucleus was also observed (i.e., 20β-hydroxy-prednisone and 20β-hydroxy-prednisolone). The yields, the absence of collateral products, and in some cases the absence of starting products allow us to say that cortisone and hydrocortisone are partly degraded.

Glucocorticoid metabolism in critically ill dogs (Canis lupus familiaris)

Critical illness due to sepsis is a major global health concern associated with a high burden of mortality and cost. Glucocorticoid dysregulation in human sepsis is associated with poorer outcomes. This study examines glucocorticoid metabolism in septic canine patients to delineate elements of cellular dysregulation in common with critically ill humans and explore potential differences. This was a prospective case–control study conducted in the veterinary specialist critical care departments of two University teaching hospitals. Critically ill canine patients with naturally occurring sepsis or septic shock were compared with an in-hospital control population. Serum total, bound, and free cortisol concentrations were increased in septic shock (P < 0.001), and higher bound cortisol was associated with nonsurvival (P = 0.026). Urinary Gas Chromatography-Tandem Mass Spectrometry was performed to assess urinary glucocorticoid metabolites and estimate intracellular glucocorticoid metabolism. Decreased renal 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) activity inferred from increased urinary cortisol-to-cortisone ratio was observed in critically ill dogs (P < 0.001). Decreased 11βHSD2 activity (P = 0.019) and increased A-ring reduction of cortisone (P = 0.001) were associated with nonsurvival within the critically ill dogs. Intriguingly, two dogs were identified with low circulating total cortisol (<2 mg/dL) associated with increased A-ring reduction of cortisol, not previously described. Investigation of spontaneous canine sepsis and septic shock reveals dysregulation of cortisol to cortisone conversion similar to that observed in human patients, but with differences in A-ring reduction compared with those reported in humans. In addition, two dogs with high levels of cortisol inactivation associated with low circulating cortisol concentrations were identified.

Glucocorticoids and regulation of brown adipose tissue in humans – physiological and pathophysiological considerations

This review discusses the effects of glucocorticoids (GCs) on brown adipose tissue (BAT) in the context of obesity prevention and therapy. Due to the unique expression of the uncoupling protein 1 (UCP1), BAT is capable of non-shivering thermogenesis, also defined as a metabolic heat production, related to increased metabolic rate. All processes that contribute to an increase in activity and/or quantity of BAT are able to upturn metabolism, and thus enable the above therapeutic goals to be achieved. GCs may stimulate BAT differentiation and proliferation. In the case of differentiation, the opposite effect of GCs has been also described. Within white adipose tissue (WAT) GCs inhibit the formation of so called beige adipocytes that are functionally and morphologically similar to the adipocytes from BAT. The activity of GCs with concomitant inhibition of WAT browning is mediated by the induction of microRNA-27b (MIR27B) expression. GCs are responsible for the decline in BAT activity as the body ages. Depriving the body of an enzyme responsible for local reduction of cortisone into an active GC-cortisol in BAT (11ß-hydroxysteroid dehydrogenase type 1; 11ß-HSD1) prevents the reduction of BAT activity. The effects of high doses of GCs on BAT generally depend on the exposure time. Prolonged elevation in GCs level decreases BAT activity. During adrenergic stimulation the effect of GCs on BAT is ambiguous, because both decrease and increase in activity has been described. A full understanding of the GCs impact on brown remodeling in WAT may reveal a discovery of a novel preventive and therapeutic strategies for obesity and possibly other metabolic disorders.

Absence of 11-keto reduction of cortisone and 11-ketotestosterone in the model organism zebrafish.

Zebrafish are widely used as model organism. Their suitability for endocrine studies, drug screening and toxicity assessements depends on the extent of conservation of specific genes and biochemical pathways between zebrafish and human. Glucocorticoids consist of inactive 11-keto (cortisone and 11-dehydrocorticosterone) and active 11β-hydroxyl forms (cortisol and corticosterone). In mammals, two 11β-hydroxysteroid dehydrogenases (11β-HSD1 and 11β-HSD2) interconvert active and inactive glucocorticoids, allowing tissue-specific regulation of glucocorticoid action. Furthermore, 11β-HSDs are involved in the metabolism of 11-oxy androgens. As zebrafish and other teleost fish lack a direct homologue of 11β-HSD1, we investigated whether they can reduce 11-ketosteroids. We compared glucocorticoid and androgen metabolism between human and zebrafish using recombinant enzymes, microsomal preparations and zebrafish larvae. Our results provide strong evidence for the absence of 11-ketosteroid reduction in zebrafish. Neither human 11β-HSD3 nor the two zebrafish 11β-HSD3 homologues, previously hypothesized to reduce 11-ketosteroids, converted cortisone and 11-ketotestosterone (11KT) to their 11β-hydroxyl forms. Furthermore, zebrafish microsomes were unable to reduce 11-ketosteroids, and exposure of larvae to cortisone or the synthetic analogue prednisone did not affect glucocorticoid-dependent gene expression. Additionally, a dual-role of 11β-HSD2 by inactivating glucocorticoids and generating the main fish androgen 11KT was supported. Thus, due to the lack of 11-ketosteroid reduction, zebrafish and other teleost fish exhibit a limited tissue-specific regulation of glucocorticoid action, and their androgen production pathway is characterized by sustained 11KT production. These findings are of particular significance when using zebrafish as a model to study endocrine functions, stress responses and effects of pharmaceuticals.

Engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to 11β-hydrocortisone.

11β-Hydrocortisone (11β-HC) is an important anti-inflammatory drug and intermediate for the synthesis of other steroids. One of the methods for the synthesis of 11β-HC is the asymmetric reduction of cortisone catalyzed by a highly regioselective and stereoselective 11β-hydroxysteroid dehydrogenase (11β-HSDH). However, this process has been prohibited by the poor soluble expression of the membrane-anchoring protein 11β-HSDH in prokaryotes. To overcome this obstacle, a mutant III-1G1 (Phe80Leu/Thr105Ser/Ala260Thr/Tyr274Stop) truncated at position 274 with improved yield of soluble protein was stepwise obtained from the 11β-HSDH from guinea pig by random mutagenesis combining with structural complementation assay and C-terminal truncating library screening. The improved 11β-HSDH mutant and glucose dehydrogenase (GDH) from Bacillus subtilis were coexpressed in Escherichia coli. The resulting whole-cell biocatalyst catalyzed the reduction of cortisone to 11β-HC with 98 % conversion in 20 h, laying foundation for the development of an asymmetric reduction process for the production of 11β-HC.

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.

Expression of 11β-hydroxysteroid dehydrogenase type 2 is deregulated in colon carcinoma.

Although the effects of glucocorticoids on proliferation, differentiation and apoptosis are well known, and steroid hormones have been identified to play a role in pathogenesis and the development of various cancers, limited data are available regarding the relationship between the local metabolism of glucocorticoids and colorectal adenocarcinoma (CRC) formation. Glucocorticoid metabolism is determined by 11β-hydroxysteroid dehydrogenases type 1 and 2 (11HSD1, 11HSD2), which increase the local concentration of cortisol due to the reduction of cortisone, or decrease this concentration due to the oxidation of cortisol. The objective of this study was to evaluate the extent of 11HSD1 and 11HSD2 mRNA in pre-malignant colorectal polyps and in CRC. The specimens were retrieved from patients by endoscopic or surgical resection and the expression of 11HSD1 and 11HSD2 was measured by real-time PCR. The polyps were of the following histological types: hyperplastic polyps and adenomas with low- or high-grade dysplasia. The neoplastic tissue of CRC obtained during tumor surgery was also studied. It was found that 11HSD2 was not only downregulated in CRC but already in the early stages of neoplastic transformation (adenoma with low-grade dysplasia). In contrast, the level of 11HSD1 was significantly increased in CRC but not in pre-malignant polyps. The results demonstrate that the downregulation of 11HSD2 gene expression is a typical feature of the development of colorectal polypous lesions and their transformation into CRC.

Regulation of glucocorticoid metabolism in the boar testis and caput epididymidis by the gonadotrophin-cAMP signalling pathway

In target tissues, cortisol is metabolised by two 11β-hydroxysteroid dehydrogenase (11βHSD) isoenzymes, namely 11βHSD1 and 11βHSD2, both of which are co-expressed in the boar testis and reproductive tract. The present study has assessed whether cortisol-cortisone metabolism in boar testis and caput epididymidis can be regulated via the gonadotrophin-cAMP signalling pathway. 11βHSD activities were measured by using a radiometric conversion assay in static tissue culture. In both testis and caput epididymidis, the net reduction of cortisone but not the net oxidation of cortisol, was significantly decreased by luteinising hormone (by 53 ± 20% and 45 ± 9%, respectively, P < 0.05), forskolin (by 60 ± 7% and 57 ± 9%, respectively, P < 0.01) and 8-bromo-cAMP (by 54 ± 4% and 64 ± 1%, respectively, P < 0.01). This suppression of 11-ketosteroid reductase activity in the boar testis by forskolin could be attenuated by the protein kinase A (PKA) inhibitor, H89. Hence, within the boar testis and the caput epididymidis, the local actions of glucocorticoids are modulated by gonadotrophin-cAMP-PKA signalling via their selective effects on the reductase activity of 11βHSD.

Inhibition of microsomal cortisol production by (–)‐epigallocatechin‐3‐gallate through a redox shift in the endoplasmic reticulum—A potential new target for treating obesity‐related diseases

Conversion of cortisone to cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in the endoplasmic reticulum (ER) of the target cells is a major determinant of glucocorticoid action, and plays an important role in the development of obesity-related diseases. Inhibition of 11βHSD1 activity is, therefore, considered as a promising novel strategy for the treatment of metabolic syndrome and diabetes. Tea flavanols and their major representative, epigallocatechin gallate are known as antiobesity and antidiabetic agents. Their impacts on blood glucose level, hepatic glucose production, and insulin responsiveness resemble those observed on inhibition or depletion of 11βHSD1. We aimed to study the effect of epigallocatechin gallate on 11βHSD1 activity in ER-derived rat liver microsomes by measuring cortisone and cortisol with HPLC. Cortisol production was efficiently suppressed in a concentration dependent manner in intact microsomal vesicles. However, this effect was abolished by membrane permeabilization; and the three proteins involved in the overall process (11βHSD1, hexose 6-phosphate dehydrogenase, and glucose 6-phosphate transporter) were not or only mildly affected. Further investigation revealed the oxidation of luminal NADPH to NADP⁺, which attenuates cortisone reduction and favors cortisol oxidation in this compartment. Such a redox shift in the ER lumen might contribute to the beneficial health effects of tea flavanols and should be regarded as a promising strategy for the development of novel selective 11βHSD1 inhibitors to treat obesity-related diseases.

Carbonyl reduction of triadimefon by human and rodent 11β-hydroxysteroid dehydrogenase 1

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11β-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11β-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11β-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11β-HSD1 inhibitor were applied to assess the role of 11β-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11β-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11β-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11β-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent Vmax/Km of recombinant human 11β-HSD1 was 5 and 15 times higher than that of mouse and rat 11β-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11β-HSD1-deficient mice indicated that 11β-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC50 values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11β-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11β-HSD1-dependent metabolism of triadimefon in humans.

Biomarkers of hypothalamic–pituitary–adrenal axis activity in mice lacking 11β-HSD1 and H6PDH

Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic–pituitary–adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11β-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11β-HSD1 is implicated in the development of obesity, and selective 11β-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11β-HSD1. We assessed biomarkers for allele combinations of 11β-HSD1 and H6PDH derived from double heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11β-HSD1 KO and double KO (DKO) adrenals were ∼30% larger than WT – indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2·2-fold higher in H6PDH KO animals and ∼1·5-fold higher in 11β-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KO of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11β-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11β-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11β-HSD1/H6PDH deficiency.

Peroxisome Proliferator–Activated Receptor-γ Activation Induces 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Human Alternative Macrophages

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator- activated receptor-γ (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPARγ therein. 11β-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPARγ induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity.

11β-Hydroxysteroid Dehydrogenase Type 1, But Not Type 2, Deficiency Worsens Acute Inflammation and Experimental Arthritis in Mice

Glucocorticoids profoundly influence immune responses, and synthetic glucocorticoids are widely used clinically for their potent antiinflammatory effects. Endogenous glucocorticoid action is modulated by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). In vivo, 11β-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11β-HSD2 catalyzes the reverse reaction, inactivating intracellular glucocorticoids. Both enzymes have been postulated to modulate inflammatory responses. In the K/BxN serum transfer model of arthritis, 11β-HSD1-deficient mice showed earlier onset and slower resolution of inflammation than wild-type controls, with greater exostoses in periarticular bone and, uniquely, ganglion cysts, consistent with greater inflammation. In contrast, K/BxN serum arthritis was unaffected by 11β-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11β-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11β-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11β-HSD1-deficient mice showed greater numbers of inflammatory cells in pleural lavages in carrageenan-induced pleurisy with lung pathology consistent with slower resolution. These data suggest that 11β-HSD1 limits acute inflammation. In contrast, 11β-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11β-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11β-hydroxysteroid dehydrogenase 1

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.

Studies on the presence of natural and synthetic corticosteroids in bovine urine

Natural and synthetic corticosteroids are widely used in veterinary medicine for their anti-inflammatory properties, but are also illegally used in animal breeding as growth-promoting agents: this latter application in livestock production has been banned within the European Union due to health concerns for the consumer. In this work urine samples collected from bovines experimentally treated with dexamethasone (0.4 mg of dexamethasone 21-disodium phosphate per capita/day for 20 consecutive days) and bovines bred under strictly controlled conditions were investigated for the presence of natural and synthetic corticosteroids, using a simple multi-residue liquid chromatography–tandem mass spectrometry method, developed and validated in accordance with the criteria of the Commission Decision 2002/657/EC. The aim of this work is to investigate the effect of a low dosage and long term dexamethasone treatment on the levels of endogenous corticosteroids in cattle and to evaluate the possible presence of prednisolone residues in bovines bred under strictly controlled conditions. Our findings confirm the high and rapid rate of dexamethasone urinary excretion. Dexamethasone treatment elicited an early reduction of hydrocortisone and cortisone, suggesting the disappearance of these two hormones as an indirect indicator of corticosteroid treatment in cattle. Prednisolone residues were found (concentration interval 0.4–1.4 ng mL −1) in urine samples collected from control bovines especially at the slaughterhouse, together with high levels of hydrocortisone and cortisone. Further studies are necessary to find out the reason of unexplained excretion of this hormone in urine samples of untreated bovines.

Cortisone-reductase deficiency associated with heterozygous mutations in 11β-hydroxysteroid dehydrogenase type 1

In peripheral target tissues, levels of active glucocorticoid hormones are controlled by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a dimeric enzyme that catalyzes the reduction of cortisone to cortisol within the endoplasmic reticulum. Loss of this activity results in a disorder termed cortisone reductase deficiency (CRD), typified by increased cortisol clearance and androgen excess. To date, only mutations in H6PD, which encodes an enzyme supplying cofactor for the reaction, have been identified as the cause of disease. Here we examined the HSD11B1 gene in two cases presenting with biochemical features indicative of a milder form of CRD in whom the H6PD gene was normal. Novel heterozygous mutations (R137C or K187N) were found in the coding sequence of HSD11B1. The R137C mutation disrupts salt bridges at the subunit interface of the 11β-HSD1 dimer, whereas K187N affects a key active site residue. On expression of the mutants in bacterial and mammalian cells, activity was either abolished (K187N) or greatly reduced (R137C). Expression of either mutant in a bacterial system greatly reduced the yield of soluble protein, suggesting that both mutations interfere with subunit folding or dimer assembly. Simultaneous expression of mutant and WT 11β-HSD1 in bacterial or mammalian cells, to simulate the heterozygous condition, indicated a marked suppressive effect of the mutants on both the yield and activity of 11β-HSD1 dimers. Thus, these heterozygous mutations in the HSD11B1 gene have a dominant negative effect on the formation of functional dimers and explain the genetic cause of CRD in these patients.

Decreased prereceptorial glucocorticoid activating capacity in starvation due to an oxidative shift of pyridine nucleotides in the endoplasmic reticulum

Redox state of pyridine nucleotides of the endoplasmic reticulum (ER) lumen was determined in different nutritional conditions. NADPH-dependent cortisone reduction and NADP +-dependent cortisol oxidation were measured in rat liver microsomes, by utilizing the luminal 11β-hydroxysteroid dehydrogenase type 1 activity. Cortisone reduction decreased, while cortisol oxidation increased during onward starvation, showing that the luminal NADPH/NADP + ratio was substantially decreased. Cortisone or metyrapone addition caused a smaller decrease in NADPH fluorescence in microsomes from starved rats. The results demonstrate that nutrient supply is mirrored by the redox state of ER luminal pyridine nucleotides.

Efficacious 11β-Hydroxysteroid Dehydrogenase Type I Inhibitors in the Diet-Induced Obesity Mouse Model

Cortisol and the glucocorticoid receptor signaling pathway have been implicated in the development of diabetes and obesity. The reduction of cortisone to cortisol is catalyzed by 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1). 2,4-Disubsituted benzenesulfonamides were identified as potent inhibitors of both the human and mouse enzymes. The lead compounds displayed good pharmacokinetics and ex vivo inhibition of the target in mice. Cocrystal structures of compounds 1 and 20 bound to human 11beta-HSD1 were obtained. Compound 20 was found to achieve high concentrations in target tissues, resulting in 95% inhibition in the ex vivo assay when dosed with a food mix (0.5 mg of drug per g of food) after 4 days. Compound 20 was efficacious in a mouse diet-induced obesity model and significantly reduced fed glucose and fasted insulin levels. Our findings suggest that 11beta-HSD1 inhibition may be a valid target for the treatment of diabetes.

HSD11B1 polymorphisms predicted bone mineral density and fracture risk in postmenopausal women without a clinically apparent hypercortisolemia

Introduction Endogenous glucocorticoid (GC) may participate in bone physiology, even in subjects with no glucocorticoid excess. 11β-hydroxysteroid dehydrogenase type 1 ( HSD11B1) is a primary regulator catalyzing the reduction of inactive cortisone to active cortisol. To elucidate genetic relevance of HSD11B1 variants to vertebral fracture and osteoporosis, we investigated the potential involvement of six HSD11B1 SNPs in postmenopausal women. Methods All exons, their boundaries and the promoter region (approximately 1.5 kb) were directly sequenced in 24 individuals. Six polymorphisms were selected and genotyped in all study participants ( n = 1329). BMD was measured using dual-energy X-ray absorptiometry. Results HSD11B1 + 16374C>T and + 27447G>C were associated with reduced vertebral fracture risk ( p = 0.016 and 0.032, respectively). Two of these (LD block2) in intron 5 (rs1000283 and rs932335) were significantly associated with bone mineral density (BMD) at the femoral neck ( p = 0.00005 and 0.0002, respectively). Specifically, HSD11B1 + 16374C>T and + 27447G>C polymorphisms were associated with higher BMD values of the femoral neck in multiple comparison ( p = 0.0002 and 0.0004, respectively) and Bonferroni corrected significance level (97% power). Consistent with these results, HSD11B1-ht21 and -ht22 comprising both SNPs also showed the evidence of association with BMD values of the femoral neck ( p domiant = 0.0002 and p recessive = 0.00005, respectively). Conclusion Our results provide preliminary evidence supporting an association of HSD11B1 with osteoporosis in postmenopausal women. Also, these findings demonstrate that + 16374C>T polymorphism may be useful genetic markers for bone metabolism.