11β-HSD1 Expression Research Articles

The expression of 11β-hydroxysteroid dehydrogenase type 1 is increased in experimental periodontitis in rats.

BackgroundThe involvement of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive glucocorticoids into active glucocorticoids intracellularly, in metabolic diseases and chronic inflammatory diseases has been elucidated. We recently reported that an increase in 11β-HSD1 expression was associated with chronic periodontitis in humans irrespective of obesity. To further clarify the role of 11β-HSD1 in chronic periodontitis, the expression of 11β-HSD1 was investigated in experimental periodontitis model in rats.MethodsExperimental periodontitis was induced by silk ligature of left maxillary second molars of 7-week-old male Wistar rats, and periodontal tissues were collected at day 3. The expression of 11β-HSD1, 11β-HSD2, and TNFα mRNA was examined using real time reverse transcription-polymerase chain reaction. The expression of TNFα was used as an indicator of inflammation. Thus, the rats in which the levels of TNFα mRNA were increased in the ligature-induced periodontitis compared with the control were analysed.ResultsThe findings demonstrated that the expression of 11β-HSD1 mRNA was significantly increased in experimental periodontitis compared with the control. The increase in the levels of 11β-HSD1 mRNA in the ligature-induced periodontitis compared with the control was positively correlated with that of TNFα mRNA. On the other hand, the expression of 11β-HSD2 mRNA, which inactivates glucocorticoids, was slightly decreased in experimental periodontitis. Therefore, the ratio of 11β-HSD1 versus 11β-HSD2 mRNA was significantly higher in experimental periodontitis than in the control.ConclusionsThese results suggest that the increased expression of 11β-HSD1, which would result in the increased levels of intracellular glucocorticoids, may play a role in the pathophysiology of experimental periodontitis.

Piper betel leaves induces wound healing activity via proliferation of fibroblasts and reducing 11β hydroxysteriod dehydrogenase-1 expression in diabetic rat

BackgroundIncreased oxidative stress and stress enzyme 11β hydroxysteriod dehydrogenase-1 (11β HSD-1) served as the major contributing factors for delayed wound healing in diabetes mellitus (DM). Piper betel (PB) leaves are reported to possess anti-diabetic, anti-oxidant and anti-microbial properties. ObjectiveThe objective was to investigate the effectiveness of topical application of PB leaves extract on oxidative stress and 11β hydroxysteriod dehydrogenase-1 (11β HSD-1) expression in diabetic wounds. Materials and methodsA total 64 male Sprague-Dawley rats were randomly chosen. The experimental rats received a single intramuscular injection of streptozotocin (45 mg/kg). Four full thickness (6 mm) wounds were created on the dorsum of each rat. The animals were equally divided (n = 8) into four groups based on the days of treatment (i.e. days 3 and 7): Control (Ctrl), diabetic untreated (DM-Ctrl), diabetic treated with 1% silver nitrate cream (DM-SN) and diabetic treated with 50 mg/kg of P. betel leaves extract (DM-PB). The rats were sacrificed on day 3 and 7 of post wound creations. ResultsFollowing day 7 wound creation, topical application of PB extract showed significant increase in hydroxyproline content, superoxide dismutase (SOD) level and decreased malondialdehyde (MDA) level, 11β-HSD-1 enzyme expression in the diabetic wounds compared to untreated diabetic wounds. The results were supported by the observations based on histological and ultrastructural features of the wound tissue applied with PB extract. ConclusionPB leaves extract improved the delayed wound healing in diabetes mellitus by decreasing the oxidative stress markers and 11β HSD-1 expression.

Elevated Hypothalamic Glucocorticoid Levels Are Associated With Obesity and Hyperphagia in Male Mice.

Glucocorticoid (Gc) excess, from endogenous overproduction in disorders of the hypothalamic-pituitary-adrenal axis or exogenous medical therapy, is recognized to cause adverse metabolic side effects. The Gc receptor (GR) is widely expressed throughout the body, including brain regions such as the hypothalamus. However, the extent to which chronic Gcs affect Gc concentrations in the hypothalamus and impact on GR and target genes is unknown. To investigate this, we used a murine model of corticosterone (Cort)-induced obesity and analyzed Cort levels in the hypothalamus and expression of genes relevant to Gc action. Mice were administered Cort (75 μg/mL) or ethanol (1%, vehicle) in drinking water for 4 weeks. Cort-treated mice had increased body weight, food intake, and adiposity. As expected, Cort increased plasma Cort levels at both zeitgeber time 1 and zeitgeber time 13, ablating the diurnal rhythm. Liquid chromatography dual tandem mass spectrometry revealed a 4-fold increase in hypothalamic Cort, which correlated with circulating levels and concentrations of Cort in other brain regions. This occurred despite decreased 11β-hydroxysteroid dehydrogenase (Hsd11b1) expression, the gene encoding the enzyme that regenerates active Gcs, whereas efflux transporter Abcb1 mRNA was unaltered. In addition, although Cort decreased hypothalamic GR (Nr3c1) expression 2-fold, the Gc-induced leucine zipper (Tsc22d3) mRNA increased, which indicated elevated GR activation. In keeping with the development of hyperphagia and obesity, Cort increased Agrp, but there were no changes in Pomc, Npy, or Cart mRNA in the hypothalamus. In summary, chronic Cort treatment causes chronic increases in hypothalamic Cort levels and a persistent elevation in Agrp, a mediator in the development of metabolic disturbances.

Compartmentalized localization of 11β-HSD 1 and 2 at the feto-maternal interface in the first trimester of human pregnancy

Glucocorticoids are engaged in a number of actions at the feto-maternal interface for the establishment of early pregnancy. However, excessive glucocorticoids can be deleterious to fetal development. Therefore, compartmentalized distribution of 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and 2), which regenerates and inactivates cortisol respectively, would ensure an optimal cortisol concentration at the feto-maternal interface for the establishment of early gestation. However, the distribution pattern of 11β-HSD1 and 2 at the feto-maternal interface in early human pregnancy is not clearly defined. Here we showed that 11β-HSD1 distributed extensively on the maternal side including decidual stromal cells and epithelial cells but scarcely on the fetal side except for localization in the fetal blood vessels of the chorionic villi. In contrast, 11β-HSD2 was abundantly localized in syncytial layer of the chorionic villi and the decidual epithelium. In primary cultures, cortisol upregulated not only 11β-HSD1 expression in decidual stromal cells but also 11β-HSD2 expression in villous trophoblasts of early pregnancy. Further studies revealed that cortisol inhibited the expression of interleukin-1β and 6 in decidual stromal cells and villous trophoblasts, and stimulated expression of human chorionic gonadotropin in villous trophoblasts. Collectively, this study has revealed a compartmentalized distribution pattern of 11β-HSD 1 and 2 at the feto-maternal interface, both of which can be upregulated by glucocorticoids, suggesting that a coordinated interaction between 11β-HSD 1 and 2 may exist to ensure an optimal cortisol concentration at discrete locations at the feto-maternal interface for the establishment of early pregnancy.

Histone methyltransferase EZH2 is critical for HSD11B2 expression in human trophoblast cells

In progressing from a simple tube to mature digestive organs, the alimentary tract is patterned along distinct axes. Transcription factors pattern the gut tube along the rostrocaudal axis, delineating a foregut, a midgut, and a hindgut. Reciprocal signals along the radial axis then enable differentiation of the mesoderm-derived mesenchyme and muscle cells from diverse endoderm-derived epithelia. Inductive interactions further specify discrete organs such as the stomach, liver, and pancreas, which show dorsoventral and left–right asymmetries. Finally, patterning of the radially symmetric small bowel along a luminal–mural axis separates progenitors in submucosal crypts from differentiated cells restricted to the villi. Diverse forces drive morphogenesis: radial cell intercalation, cell shape changes, asymmetric tissue growth, and tension from surrounding structures. Much remains unclear about how positional and inductive cues activate particular genes to implement this developmental program.

Expression and regulation of 11β-hydroxysteroid dehydrogenase type 1 in first trimester human decidua cells: Implication in preeclampsia

Glucocorticoids are implicated in successful blastocyst implantation, whereas alterations in glucocorticoid levels are associated with various pregnancy disorders including preeclampsia. Tissue concentration of active glucocorticoids depends on the expression of 11β-hydroxysteroid dehydrogenase (11β-HSD). This study investigated the contribution of first trimester decidua to glucocorticoid availability at the fetal-maternal interface by assessing the expression and regulation of 11β-HSD in human first trimester decidual tissues and cells and by evaluating 11β-HSD levels in preeclamptic vs. gestational age-matched decidua. 11β-HSD1 was the predominant isoform in first trimester decidua. In vitro, decidual cell 11β-HSD1 levels and enzymatic activity were up-regulated by ovarian steroids and inflammatory cytokines. Higher levels of 11β-HSD1 were found in preeclamptic decidua compared to controls. The present study indicates the predominance of 11β-HSD oxoreductase isoform in early decidua. Observations that ovarian hormones and inflammatory cytokines up-regulate 11β-HSD1, together with increased 11β-HSD1 expression in preeclampsia, highlight a role for decidual cells in controlling biologically active glucocorticoids in early pregnancy.

Inhibition of placental 11beta-hydroxysteroid dehydrogenase type 2 by lead.

Lead interferes with cortisol blood concentration, increases the risk of obstetrical complications, and could alter fetal development. The placenta controls maternal cortisol transfer to the fetus by the activity of the type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2), which converts cortisol into inactive cortisone. This study determines the effect of lead on the expression and activity of the placental 11β-HSD2 in human trophoblast-like BeWo cells. Cells were treated with increasing concentration (0-1000nM) of PbCl2 for 24h. 11β-HSD2 protein expression was reduced by 45% at 1000nM of PbCl2 compared to untreated cells, while the activity was significantly reduced by PbCl2 at 10, 100 and 1000nM. This study shows the direct inhibitory action of lead on placental 11β-HSD2 activity and suggests that this heavy metal reduces the efficiency of the placental protection against the adverse effects of high cortisol level during fetal development.

Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS)-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients.

Up-regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 Expression by Hedgehog Ligand Contributes to the Conversion of Cortisol Into Cortisone.

The cortisol-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that catalyzes the intracellular inactivation of glucocorticoids plays a pivotal role in human pregnant maintenance and normal fetal development. Given the fact that the main components of Hedgehog (HH) signaling pathway are predominantly expressed in syncytial layer of human placental villi where 11β-HSD2 is robustly expressed, in the present study, we have investigated the potential roles and underlying mechanisms of HH signaling in 11β-HSD2 expression. Activation of HH signaling by a variety of approaches robustly induced 11β-HSD2 expression as well as the 11β-HSD2 activity, whereas suppression of HH signaling significantly attenuated 11β-HSD2 expression as well as the 11β-HSD2 activity in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in HH signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated HH-induced 11β-HSD2 expression and activity, and overexpression of GLI2 activator alone was sufficient to induce 11β-HSD2 expression and activity. Finally, GLI2 not only directly bound to the promoter region of gene hsd11b2 to transactivate hsd11b2 but also formed a heterodimer with RNA polymerase II, an enzyme that catalyzes the transcription of DNA to synthesize mRNAs, resulting in up-regulation of hsd11b2 gene transcription. Taken together, the present study has uncovered a hitherto uncharacterized role of HH/GLI2 signaling in 11β-HSD2 regulation, implicating that HH signaling through GLI2 could be required for the human pregnant maintenance and fetal development.

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 ameliorates obesity-related insulin resistance

Excess 11β–hydroxysteroid dehydrogenase type 1 (11β-HSD1) may be implicated in the development of obesity related metabolic disorders. The present study measured the expression level of 11β-HSD1 in visceral adipose tissues from 23 patients undergoing abdominal operation. Correlation of 11β-HSD1 expression with BMI, waist-to-hip ratio (WHR), HOMA-IR, and serum lipids was evaluated by spearman correlation analysis. High-fat diet-induced obese (DIO) rats were orally dosed with BVT.2733 for 4 weeks. Weight, plasma insulin, and lipids were detected at the end of the treatment. The effects of 11β-HSD1 inhibition on the key insulin-signaling cascade and adipocytokines were measured by western blot and ELISA respectively. 11β-HSD1 was increased in patients with central obesity, the expression level of which was closely related with WHR (r = 0.5851), BMI (r = 0.4952), and HOMA-IR (r = 0.4637). Obesity related insulin resistance in high-fat DIO rats, as reflected by a marked decrease in IRS-1, IRS-2, GLUT4, and PI3K, could be attenuated by 11β-HSD1 inhibition. Furthermore, the down-regulation of 11β-HSD1 could correct the disordered profiles of adipocytokines including adiponectin, IL-6, and TNF-α. These findings indicated that 11β-HSD1 inhibition can give a potential benefit in reducing obesity and lowering insulin resistance by modulating the insulin-signaling pathway and adipocytokine production.

Novel small molecule 11β-HSD1 inhibitor from the endophytic fungus Penicillium commune.

Two new phenone derivatives penicophenones A (1) and B (2), a new cyclic tetrapeptide penicopeptide A (3), and five known compounds were isolated from the culture broth of Penicillium commune, an endophytic fungus derived from Vitis vinifera. Compounds 1–3 were elucidated by extensive spectroscopic analyses including 1D and 2D NMR and HRESIMS. The absolute configurations of 1 and 3 were determined by comparing its ECD with related molecules and modified Marfey’s analysis, respectively. Penicophenone A (1) possesses a rare benzannulated 6,6-spiroketal moiety, which is a new member of the unusual structural class with peniphenone A as the representative. Compound 3 exhibited significant inhibition activities against 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in vitro and showed strong binding affinity to 11β-HSD1. Moreover, compound 3 treatments decreased the lipid droplet accumulation associate with the inhibition of 11β-HSD1 expression in differentiate-induced 3T3-L1 preadipocytes. Furthermore, the molecular docking demonstrated that compound 3 coordinated in the active site of 11β-HSD1 is essential for the ability of diminishing the enzyme activity.

11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice.

Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11β-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11β-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11β-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b+,Ly6G+,7/4+ cells) as the thioglycollate-recruited cells that most highly express 11β-HSD1 and show dynamic regulation of 11β-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11β-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11β-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11β-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11β-HSD1, acute inhibition of 11β-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11β-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11β-HSD1 expression, suggesting the antiinflammatory effects of 11β-HSD1 in neutrophils may be conserved in humans.

Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11β-hydroxysteroid dehydrogenase type 1

Background and aimsData about glucocorticoids role in the development of atherosclerosis are controversial showing different effects in human than in experimental animal models. Atherosclerosis is the result of a chronic inflammatory response to an injured endothelium where an uncontrolled uptake of OxLDL by macrophages triggers the development of foam cells, the main component of fatty streaks in atherosclerotic plaque. There are few data about the direct effect of glucocorticoids in macrophages of atherosclerotic plaque. The aim of the study was to elucidate the role of glucocorticoids in the development of foam cells in atherosclerosis initiation. MethodsFor this purpose we used THP1 cells differentiated to macrophages with phorbol esters and incubated with OxLDL alone or with cortisol or cortisone. THP1 cells were also incubated with cortisone plus an inhibitor of 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) activity to determine the role of this enzyme on glucocorticoid action in this process. ResultsOurs results showed that cortisol and cortisone decreased significantly the inflammation promoted by OxLDL, and also diminished the expression of genes involved in influx and efflux of cholesterol resulting in a reduced lipid accumulation. Likewise cortisol and cortisone decreased 11βHSD1 expression in THP1 cells. The presence of the inhibitor of 11βHSD1 abolished all the effects elicited by cortisone. ConclusionOur results indicate a direct effect of glucocorticoids on macrophages braking atherosclerosis initiation, reducing pro-inflammatory markers and OxLDL uptake and cholesterol re-esterification, but also inhibiting cholesterol output. These effects appear to be mediated, at least in part, by 11βHSD1 activity.

Neuroendocrinological and Epigenetic Mechanisms Subserving Autonomic Imbalance and HPA Dysfunction in the Metabolic Syndrome.

Impact of environmental stress upon pathophysiology of the metabolic syndrome (MetS) has been substantiated by epidemiological, psychophysiological, and endocrinological studies. This review discusses recent advances in the understanding of causative roles of nutritional factors, sympathomedullo-adrenal (SMA) and hypothalamic-pituitary adrenocortical (HPA) axes, and adipose tissue chronic low-grade inflammation processes in MetS. Disturbances in the neuroendocrine systems for leptin, melanocortin, and neuropeptide Y (NPY)/agouti-related protein systems have been found resulting directly in MetS-like conditions. The review identifies candidate risk genes from factors shown critical for the functioning of each of these neuroendocrine signaling cascades. In its meta-analytic part, recent studies in epigenetic modification (histone methylation, acetylation, phosphorylation, ubiquitination) and posttranscriptional gene regulation by microRNAs are evaluated. Several studies suggest modification mechanisms of early life stress (ELS) and diet-induced obesity (DIO) programming in the hypothalamic regions with populations of POMC-expressing neurons. Epigenetic modifications were found in cortisol (here HSD11B1 expression), melanocortin, leptin, NPY, and adiponectin genes. With respect to adiposity genes, epigenetic modifications were documented for fat mass gene cluster APOA1/C3/A4/A5, and the lipolysis gene LIPE. With regard to inflammatory, immune and subcellular metabolism, PPARG, NKBF1, TNFA, TCF7C2, and those genes expressing cytochrome P450 family enzymes involved in steroidogenesis and in hepatic lipoproteins were documented for epigenetic modifications.

Local Glucocorticoid Activation by 11β-Hydroxysteroid Dehydrogenase 1 in Keratinocytes: The Role in Hapten-Induced Dermatitis

Over the past decade, extra-adrenal cortisol production was reported in various tissues. The enzyme thatcatalyzes the conversion of hormonally inactive cortisone into active cortisol in cells is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is also expressed in keratinocytes and regulates inflammation and keratinocyte proliferation. To investigate the function of 11β-HSD1 in keratinocytes during inflammation invivo, we created keratinocyte-specific 11β-HSD1 knockout (K5-Hsd11b1-KO) mice and analyzed the inflammatory response in models of hapten-induced contact irritant dermatitis. K5-Hsd11b1-KO mice showed enhanced ear swelling in low-dose oxazolone-, 2,4,6-trinitro-1-chlorobenzene (TNCB)-, and 2,4-dinitrofluorobenzene-induced irritant dermatitis associated with increased inflammatory cell infiltration. Topical application of corticosterone dose dependently suppressed TNCB-induced ear swelling and cytokine expression. Similarly in mouse keratinocytes invitro, corticosterone dose dependently suppressed 2,4,6-trinitrobenzenesulfonic acid-induced IL-1α and IL-1β expression. The effect of 11-dehydrocorticosterone was attenuated in TNCB-induced irritant dermatitis in K5-Hsd11b1-KO mice compared with wild-type mice. In human samples, 11β-HSD1 expression was decreased in epidermis of psoriasis vulgaris compared with healthy skin. Taken together, these data suggest that corticosterone activation by 11β-HSD1 in keratinocytes suppresses hapten-induced irritant dermatitis through suppression of expression of cytokines, such as IL-1α and IL-1β, in keratinocytes.

Potential role of renal mineralocorticoid receptor increasing the blood pressure sensitivity in female mice exposed to early life stress (ELS)

Epidemiological studies have shown that exposure to ELS increases the number of risk factors to develop cardiovascular and metabolic disease later in life. Previously, we have shown that C57BL/6 mice exposed to maternal separation (MatSep), a model of ELS, display exaggerated diet‐induced obesity. However, only female MatSep mice show increased visceral fat and glucose intolerance compared to male MatSep mice. In addition, we have reported increased local glucocorticoid regeneration in liver and adipose tissue as a potential mechanism of ELS‐induced obesity. The aim of this study was to investigate the impact of MatSep in combination with a high fat diet (HFD) on blood pressure and renal gene expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and 11 β‐hydroxysteroid dehydrogenase types 1 and 2. MatSep was performed in C57BL/6 mice pups by daily separation from the dam during the first two weeks of life. Non‐disturbed litters were used as controls. Upon weaning, mice were fed a HFD (60% kcal from fat) for 16 weeks. Female mice were implanted with radiotelemetry at week 14 (DSI, n=4–5). Whole kidney tissue from female mice was collected and snap frozen for gene expression determination using RT‐PCR (n=6–8). Mean arterial pressure (24hr average) was elevated in female MatSep mice compared to control (133.1±0.1 vs. 122.8±0.7 mmHg, p<0.05). Pulse pressure was increased in MatSep mice as well (35.1±0.1 vs. 31.4±0.4 mmHg, p<0.05). No differences were observed in HR (622.2±21.6 vs. 638.1±28.5 bpm, p<0.05); however, spontaneous baroreflex sensitivity was reduced in female mice exposed to MatSep (1.67±0.18 vs. 2.52±0.24 msec/mmHg, p<0.05). Renal expression levels of 11βHSD1 were similar in female MatSep and control mice fed a HFD. Yet, 11βHSD2 mRNA expression was attenuated in female MatSep mice compared to control mice (0.6±0.1 vs. 1.0±0.1 fold, p<0.05). GR expression was similar between groups but MR expression was reduced in MatSep mice vs. control (0.5±0.1 vs. 1.0±0.1 fold, respectively, p<0.05). Additional experiments revealed that MatSep had no effect on renal 11βHSD1 or 11βHSD2 mRNA expression in male mice. Furthermore, no differences in gene expression were observed between MatSep and control groups when the mice were fed a low fat diet. Interestingly, estrogen receptor β (ERβ) mRNA expression in the kidney was significantly upregulated in obese female MatSep mice (3.8±0.3 vs. 1.1±0.2 fold, respectively, p<0.05), with no differences in ERα expression. Since the analysis of the 11βHSD2 promoter revealed 4 estrogen response elements ~50,000 bp near to its start point, further investigation in the control of this enzyme by estradiol will be conducted. Taken together, these data suggest that in response to a second hit such as HFD, MatSep may induce MR overstimulation leading to increases in blood pressure sensitivity in female mice.Support or Funding InformationNIH R00 HL111354; COBRE P20 GM103527‐06

The secretion, synthesis, and metabolism of cortisol and its downstream genes in the H–P–I axis of rare minnows (Gobiocypris rarus) are disrupted by acute waterborne cadmium exposure

The H (hypothalamic)–P (pituitary)–I (interrenal) axis plays a critical role in the fish stress response and is regulated by several factors. Cadmium (Cd) is one of the most toxic heavy metals in the world, but its effects on the H–P–I axis of teleosts are largely unknown. Using rare minnow (Gobiocypris rarus) as an experimental animal, we found that Cd only disrupted the secretion and synthesis of cortisol. Neither hormones at the H or P level nor the expressions of their receptor genes (corticotropin-releasing hormone receptor (CRHR) and melanocortin receptor 2 (MC2R)) were affected. Steroidogenic acute regulator (StAR), CYP11A1 and CYP11B1, which encode the key enzymes in the cortisol synthesis pathway, were significantly up-regulated in the kidney (including the head kidney). The level of 11β-HSD2, which is required for the conversion of cortisol to cortisone, was increased in the kidney, intestine, brain, and hepatopancreas, whereas the expression of 11β-HSD1, which encodes the reverse conversion enzyme, was increased in the gill, kidney and almost unchanged in other tissues. The enzyme activity concentration of 11β-HSD2 was increased in the kidney as well. The level of glucocorticoid receptor (GR) decreased in the intestine, gill and muscle, and the key GR regulator FK506 binding protein5 (FKBP5) was up-regulated in the GR-decreased tissues, whereas the level of nuclear receptor co-repressor 1 (NCoR1), another GR regulator remained almost unchanged. Thus, GR, FKBP5 and 11β-HSD2 may be involved in Cd-induced cortisol disruption.

Diploid and triploid African catfish (Clarias gariepinus) differ in biomarker responses to the pesticide chlorpyrifos

The impacts of environmental stressors on polyploid organisms are largely unknown. This study investigated changes in morphometric, molecular, and biochemical parameters in full-sibling diploid and triploid African catfish (Clarias gariepinus) in response to chlorpyrifos (CPF) exposures. Juvenile fish were exposed to three concentrations of CPF (mean measured μg/L (SD): 9.71 (2.27), 15.7 (3.69), 31.21 (5.04)) under a static-renewal condition for 21days. Diploid control groups had higher hepatosomatic index (HSI), plasma testosterone (T), and brain GnRH and cyp19a2 expression levels than triploids. In CPF-exposed groups, changes in HSI, total weight and length were different between the diploid and triploid fish. In contrast, condition factor did not alter in any of the treatments, while visceral-somatic index (VSI) changed only in diploids. In diploid fish, exposure to CPF did not change brain 11β-hsd2, ftz-f1, foxl2, GnRH or cyp19a2 mRNA levels, while reduced tph2 transcript levels compared to the control group. In contrast, 11β-hsd2 and foxl2 expression levels were changed in triploids following CPF exposures. In diploids, plasma T levels showed a linear dose-response reduction across CPF treatments correlating with liver weight and plasma total cholesterol concentrations. In contrast, no changes in plasma cholesterol and T concentrations were observed in triploids. Plasma cortisol and 17-β estradiol (E2) showed no response to CPF exposure in either ploidy. Results of this first comparison of biomarker responses to pesticide exposure in diploid and polyploid animals showed substantial differences between diploid and triploid C. gariepinus.

Yeast-based assays for screening 11β-HSD1 inhibitors.

BackgroundIntracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11β-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11β-HSD1 inhibitors.ResultsThe yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11β-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11β-HSD1 inhibitory activity. As proof of concept the correlation between 11β-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11β-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11β-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11β-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times.ConclusionsOur yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11β-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11β-HSD1 inhibitors at intracellular level.

Local Regeneration of Cortisol by 11β-HSD1 Contributes to Insulin Resistance of the Granulosa Cells in PCOS.

Insulin resistance (IR) of the granulosa cells may account for the ovarian dysfunctions observed in polycystic ovarian syndrome (PCOS). The underlying mechanism remains largely unresolved. The objective of the study was to investigate the relationship of IR of the granulosa cells with cortisol in the follicular fluid and 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and -2) in the granulosa cells in PCOS. Follicular fluid and granulosa cells were collected from non-PCOS and PCOS patients with and without IR to measure cortisol concentration and the amounts of 11β-HSD1 and -2, which were then correlated with IR status. The effects of cortisol on the expression of genes pertinent to IR were studied in cultured human granulosa cells. Cortisol concentration in the follicular fluid, 11β-HSD1 but not 11β-HSD2 mRNA in the granulosa cells were significantly elevated in PCOS with IR. Increased reductase and decreased oxidase activities of 11β-HSD were observed in granulosa cells in PCOS with IR. In cultured granulosa cells, insulin-induced Akt phosphorylation was significantly attenuated by cortisol. Cortisol not only increased phosphatase and tensin homolog deleted on chromosome 10, an inhibitor of Akt phosphorylation, but also 11β-HSD1 in the cells. Increased 11β-HSD1 expression and its reductase activity in granulosa cells are the major causes of increased cortisol concentration in the follicular fluid of PCOS with IR. The consequent excessive cortisol might contribute to IR of the granulosa cells in PCOS patients by attenuating Akt phosphorylation via induction of phosphatase and tensin homolog deleted on chromosome 10 expression, which might be further exacerbated by the induction of 11β-HSD1.