Glucocorticoid action within the cells is regulated by the levels of glucocorticoid receptor (GR) expression and two enzymes, 11-beta hydroxysteroid dehydrogenase type 1 (11betaHSD1), which converts inactive to active glucocorticoids, and 11-beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), which regulates the access of active glucocorticoids to the receptor by converting cortisol/corticosterone to the glucocorticoid-inactive form cortisone/dehydrocorticosterone. Male Wistar rats developed obesity by being fed a high-fat diet for 56 days, and GR, 11betaHSD1 and 11betaHSD2 gene expression were compared with control-diet fed animals. Gene expression analysis of 11betaHSD1, 11betaHSD2 and GR were performed by RT-PCR in subcutaneous and retroperitoneal adipose tissue. High-fat fed animals overexpressed 11betaHSD2 in subcutaneous but not in retroperitoneal fat. Interestingly, mRNA levels strongly correlated in both tissues with different parameters related to obesity, such as body weight, adiposity and insulin resistance, suggesting that this gene is a reliable marker of adiposity in this rat model of obesity. Thus, 11betaHSD2 is expressed in adipose tissue by both adipocytes and stromal-vascular cells, which suggests that this enzyme may play an important role in preventing fat accumulation in adipose tissue.

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.

Exposure to maternal cortisol plays a crucial role in fetal organogenesis. However, fetal overexposure to cortisol has been linked to a range of short- and long-term adverse outcomes. Normally, this is prevented by the expression of an enzyme in the placenta called 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) which converts active cortisol to its inactive metabolite cortisone. Placental 11β-HSD2 is known to be reduced in a number of adverse pregnancy complications, possibly through an epigenetic mechanism. As a result, a number of pan-HDAC inhibitors have been examined for their ability to promote 11β-HSD2 expression. However, it is not known if the effects of pan-HDAC inhibition are a general phenomenon or if the effects are dependent upon a specific class of HDACs. Here, we examined the ability of pan- and class-specific HDAC inhibitors to regulate 11β-HSD2 expression in JEG3 cells. We find that pan-, class I, or class IIa HDAC inhibition promoted 11β-HSD2 expression and prevented cortisol or interleukin-1β-induced decrease in its expression. These results demonstrate that targeting a specific class of HDACs can promote 11β-HSD2 expression in JEG3 cells. This adds to the growing body of evidence suggesting that HDACs may be crucial in maintaining normal fetal development.

11-Beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) converts inactive cortisone into active cortisol, thereby amplifying intracellular glucocorticoid action. The efficacy and safety of the 11betaHSD1 inhibitor INCB13739 were assessed when added to ongoing metformin monotherapy in patients with type 2 diabetes exhibiting inadequate glycemic control (A1C 7-11%).

Glucocorticoid hormones play a major role in fetal organ maturation. Yet, excessive glucocorticoid exposure in utero can result in a variety of detrimental effects, such as growth retardation and increased susceptibility to the development of hypertension. To protect the fetus, maternal glucocorticoids are metabolized into inactive compounds by placental 11beta-hydroxysteroid dehydrogenase type2 (11βHSD2). This enzyme is also expressed in the kidney, where it prevents illicit occupation of the mineralocorticoid receptor by glucocorticoids. We investigated the role of renal 11βHSD2 in the control of neonatal glucocorticoid metabolism in the human and mouse.

Small for gestational age infants have greater risk of developing metabolic diseases in adult life. It has been suggested that low birth weight may result from glucocorticoid excess in utero, a key mechanism in fetal programming. The placental enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2, HSD11B2 gene) acts as a barrier protecting the fetus from maternal corticosteroid deleterious effects. Low placental 11β-HSD2 transcription and activity have been associated with low birth weight, yet the mechanism regulating its protein expression is not fully understood. In the present study we aimed to analyze 11β-HSD2 protein expression in placentas of adequate and small for gestational age (AGA and SGA, respectively) newborns from healthy mothers, and to explore whether 11β-HSD2 protein expression could be modulated by DNA methylation. 11β-HSD2 protein levels were measured by western blot in placental biopsies from term AGA and SGA infants (n=10 per group). DNA methylation was profiled both globally and in the HSD11B2 promoter by liquid chromatography with UV detection and methylation-specific melting curve analysis, respectively. We found lower placental 11β-HSD2 protein expression and higher HSD11B2 promoter methylation in SGA compared to AGA. Promoter methylation was inversely correlated with both protein expression and, importantly, birth weight. No changes in global placental methylation were found. In conclusion, lower 11β-HSD2 protein expression is associated with higher HSD11B2 promoter methylation, correlating with birth weight in healthy pregnancy. Our data support the role of 11β-HSD2 in determining birth weight, providing evidence of its regulation by epigenetic mechanisms, which may affect postnatal metabolic disease risk.

Mineralocorticoid-receptor (MR) dysfunction as expressed by low systolic blood pressure and a high salivary aldosterone/cortisol ratio predicts less favorable antidepressant treatment outcome. Inhibition of peripheral 11-beta-hydroxysteroid-dehydrogenase type 2 (11betaHSD2) reverses these markers. We therefore tested the hypothesis that the 11betaHSD2 inhibitor glycyrrhizin affects treatment outcome via this mechanism. We administered Glycyrrhiza glabra (GG) extract containing 7-8 % of glycyrrhizin at a dose of 2 × 700 mg daily adjunct to standard antidepressants in hospitalized patients with major depression. These subjects were compared in an open-label fashion with patients, who did not receive GG (treatment as usual, TAU). Assessments were done at baseline and approximately 2 weeks after. Twelve subjects were treated with GG and compared to 55 subjects with TAU. At week 2, the Hamilton Depression Rating Scale (HAMD-21) change from baseline as well as the CGI-S change showed a significant time × treatment interaction (p < 0.03), indicating a possible therapeutic benefit of GG. Clinical benefit seems to be more pronounced in subjects with lower systolic blood pressure and significantly correlated with reduced sleep duration in the GG group. Our preliminary data show that treatment with the 11betaHSD2 inhibitor glycyrrhizin may possess a beneficial effect on antidepressant response, which may be specific to a defined depression subtype.

To study the oral 11 beta-hydroxysteroid dehydrogenase-1 (11β-HSD1) inhibitor BI 187004 (NCT02150824), as monotherapy and in combination with metformin, versus placebo in patients with type 2 diabetes mellitus (T2DM) affected by overweight or obesity.

Metabolic syndrome is considered the precursor of type 2 diabetes mellitus. Tuberculosis is a leading infection that constitutes a global threat remaining a major cause of morbi-mortality in developing countries. People with type 2 diabetes mellitus are more likely to suffer from infection with Mycobacterium tuberculosis. For both type 2 diabetes mellitus and tuberculosis, there is pulmonary production of anti-inflammatory glucocorticoids mediated by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The adrenal hormone dehydroepiandrosterone (DHEA) counteracts the glucocorticoid effects of cytokine production due to the inhibition of 11β-HSD1. Late advanced tuberculosis has been associated with the suppression of the Th1 response, evidenced by a high ratio of cortisol/DHEA. In a murine model of metabolic syndrome, we determined whether DHEA treatment modifies the pro-inflammatory cytokines due to the inhibition of the 11β-HSD1 expression. Since macrophages express 11β-HSD1, our second goal was incubating them with DHEA and Mycobacterium tuberculosis to show that the microbicide effect was increased by DHEA. Enoyl-acyl carrier protein reductase (InhA) is an essential enzyme of Mycobacterium tuberculosis involved in the mycolic acid synthesis. Because 11β-HSD1 and InhA are members of a short-chain dehydrogenase/reductase family of enzymes, we hypothesize that DHEA could be an antagonist of InhA. Our results demonstrate that DHEA has a direct microbicide effect against Mycobacterium tuberculosis; this effect was supported by in silico docking analysis and the molecular dynamic simulation studies between DHEA and InhA. Thus, DHEA increases the production of pro-inflammatory cytokines in the lung, inactivates GC by 11β-HSD1, and inhibits mycobacterial InhA. The multiple functions of DHEA suggest that this hormone or its synthetic analogs could be an efficient co-adjuvant for tuberculosis treatment.

Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate.

The metabolic syndrome is defined by impaired carbohydrate metabolism and lipid disorders and often accompanied by hypertension, all of which will lead to obesity and insulin resistance. Glucocorticoids play a regulatory role in the metabolism of proteins, lipids, and carbohydrates. There is growing evidence for a role of glucocorticoids in the development of the metabolic syndrome. The most important factor that regulates the access of endogenous glucocorticoids to receptors after release of glucocorticoids and their diffusion into the cytoplasm of target cells is the steroid metabolism involving a microsomal enzyme, 11β-hydroxysteroid dehydrogenase (11β-HSD). The changes in intracellular glucocorticoid metabolism in the pathogenesis of obesity indicate the participation of modulation by 11β-HSD1, which may represent a new therapeutic target for the treatment of diseases such as type 2 diabetes, visceral obesity, or atherosclerosis. The aim of our study was to determine the fast and effective method to assess inhibition activity of compounds in relation with 11β-hydroxysteroid dehydrogenase. The material for this study was human liver and kidney microsomes. In this study we used ELISA technique using 96-well microplates coated with antibodies which were specific for analyzed enzymes. The method can quickly and efficiently measure the inhibition of both 11β-HSD1 and 11β-HSD2. This method can be used to search for and determine inhibitors of this enzyme. Cortisone and cortisol were used as the substrates for corresponding enzyme assays. Furthermore, 3-N-allyl-2-thiouracil derivatives were used by us for comparison purposes in developing the method, although, due to their structure, those derivatives have not previously been considered as potential inhibitors of 11β-HSD1. 3-N-Allyl-2-thiouracil derivatives are a group worth considering, because by modifying their structure (e.g., by introducing other substituents into the pyrimidine ring) it will be possible to obtain an increase in the activity of compounds in this regard. In conclusion, this study shows an efficient and fast method of determining inhibition activity of compounds in relation with 11β-hydroxysteroid dehydrogenase.

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) regulates tissue-specific glucocorticoid metabolism and its impaired expression and activity are associated with major diseases. Pharmacological inhibition of 11β-HSD1 is considered a promising therapeutic strategy. This study investigated whether alternative 7-oxo bile acid substrates of 11β-HSD1 or the ratios to their 7-hydroxy products can serve as biomarkers for decreased enzymatic activity.

Pulmonary tuberculosis (PTB), caused by Mycobacterium tuberculosis (Mtb), is a major health problem worldwide, further aggravated by the convergence of type 2 diabetes mellitus (DM) which constitutes an important risk factor for TB development. The worse scenario of patients with PTB and DM may be partly related to a more unbalanced defensive response. As such, newly diagnosed PTB patients with DM (TB+DM, n = 11) or not (TB, n = 21), as well as DM (n = 18) patients and pair matched controls (Co, n = 22), were investigated for the circulating immuno-endocrine-metabolic profile (ELISA), along with studies in peripheral blood mononuclear cells (PBMC) analyzing transcript expression (RT-qPCR) of mediators involved in glucocorticoid functionality. Given the hyperglycemic/hypercortisolemic scenario of TB+DM patients, PBMC were also exposed to stress-related cortisol concentrations (0.1 and 1 μM) and supraphysiologic glucose doses (10, 20, and 40 mM) and assessed for the specific response against Mtb stimulation (lymphoproliferation, -thymidine incorporation-, and cytokine production -bead-cytometry). All TB patients displayed increased plasma amounts of cortisol, growth hormone -hGH-, and proinflammatory mediators. In turn, TB+DM showed even higher levels of interferon gamma -IFN-γ- and hGH (vs. TB), or IL-6, C reactive protein, cortisol and hGH (vs. DM). Both DM groups had equally augmented values of IL-10. All TB patients showed decreased dehydroepiandrosterone- sulfate concentrations, even more in TB+DM cases. Leptin was also decreased in both TB cases, particularly in the TB group, revealing a lower body mass index, as well. Unlike PBMC from TB cases showing a decreased relationship between the glucocorticoids receptor (GR) isoforms (GRα/GRβ; functional isoform/negative isoform), cells from TB+DM patients had no changes in this regard, along with an increased expression of 11-beta hydroxysteroid dehydrogenase type-1, the enzyme facilitating intracellular cortisone to cortisol conversion. TB+DM patients also showed an increased Mtb antigen-driven lymphoproliferation. Compared to TB, DM and HCo counterparts, PBMC from TB+DM patients had a biased Th1 response to Mtb stimulation (increased IL-2 and IFN-γ production), even when exposed to inhibitory cortisol doses. TB+DM patients show a more unbalanced immuno-endocrine relationship, respect the non-diabetic counterparts, with a relative deficiency of cortisol immunomodulatory influences, despite their more favorable microenvironment for cortisol-mediated immune effects.

Glucocorticoid (GC) hormones act on the brain to regulate diverse functions, from behavior and homeostasis to the activity of the hypothalamic-pituitary-adrenal axis. Local regeneration and metabolism of GCs can occur in target tissues through the actions of the 11β-hydroxysteroid dehydrogenases [11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), respectively] to regulate access to GC receptors. Songbirds have become especially important model organisms for studies of stress hormone action; however, there has been little focus on neural GC metabolism. Therefore, we tested the hypothesis that 11β-HSD1 and 11β-HSD2 are expressed in GC-sensitive regions of the songbird brain. Localization of 11β-HSD expression in these regions could provide precise temporal and spatial control over GC actions. We quantified GC sensitivity in zebra finch (Taeniopygia guttata) brain by measuring glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) expression across six regions, followed by quantification of 11β-HSD1 and 11β-HSD2 expression. We detected GR, MR, and 11β-HSD2 mRNA expression throughout the adult brain. Whereas 11β-HSD1 expression was undetectable in the adult brain, we detected low levels of expression in the brain of developing finches. Across several adult brain regions, expression of 11β-HSD2 covaried with GR and MR, with the exception of the cerebellum and hippocampus. It is possible that receptors in these latter two regions require direct access to systemic GC levels. Overall, these results suggest that 11β-HSD2 expression protects the adult songbird brain by rapid metabolism of GCs in a context and region-specific manner.

The nucleus accumbens (NAc) is part of a forebrain system implicated in reward, motivation, and learning. NAc neurons become activated during various ingestive activities, including salt intake. A subset of neurons within the nucleus tractus solitarius (NTS) shows c-Fos activation during prolonged sodium deprivation in rats. These neurons express mineralocorticoid receptors and the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them selectively sensitive to aldosterone-an adrenal hormone that modulates sodium appetite. Here we tested whether these neurons project multisynaptically to the core or shell subregions of the NAc. Pseudorabies virus (PRV)-a retrograde transneuronal tracer-was injected into the NAc in rats and after 3-4 days PRV-infected HSD2 neurons were identified. PRV injections into the NAc core yielded greater numbers of PRV-labeled HSD2 neurons than did comparable injections into the NAc shell. Transneuronal labeling was also found in brainstem sites that receive direct projections from HSD2 neurons, namely, lateral parabrachial and prelocus coeruleus nuclei. In other experiments a retrograde neural tracer (cholera toxin beta-subunit) was injected into the NAc. Extensive retrograde labeling was found in the midline thalamus and frontal cortical regions, but no cells were labeled in the NTS or parabrachial region. These findings indicate that the HSD2 neurons project via a multisynaptic pathway to the NAc, which may be relayed sequentially through two sites: the dorsolateral pons and the paraventricular thalamic nucleus. HSD2 neurons may be part of an ascending pathway involved in the salt-seeking behavior of sodium-depleted rats.

The nucleus of the solitary tract (NTS) contains a subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them uniquely sensitive to aldosterone. These neurons may drive sodium appetite, which is enhanced by aldosterone. Anterograde and retrograde neural tracing techniques were used to reveal the efferent projections of the HSD2 neurons in the rat. First, the anterograde tracer Phaseolus vulgaris leucoagglutinin was used to label axonal projections from the medial NTS. Then, NTS-innervated brain regions were injected with a retrograde tracer, cholera toxin beta subunit, to determine which sites are innervated by the HSD2 neurons. The HSD2 neurons project mainly to the ventrolateral bed nucleus of the stria terminalis (BSTvl), the pre-locus coeruleus (pre-LC), and the inner division of the external lateral parabrachial nucleus (PBel). They also send minor axonal projections to the midbrain ventral tegmental area, lateral and paraventricular hypothalamic nuclei, central nucleus of the amygdala, and periaqueductal gray matter. The HSD2 neurons do not innervate the ventrolateral medulla, a key brainstem autonomic site. Additionally, our tracing experiments confirmed that the BSTvl receives direct axonal projections from the neighboring A2 noradrenergic neurons in the NTS, and from the same pontine sites that receive major inputs from the HSD2 neurons (PBel and pre-LC). The efferent projections of the HSD2 neurons may provide new insights into the brain circuitry responsible for sodium appetite.

Glucocorticoid therapy is the most common cause of iatrogenic osteoporosis. Less is known regarding the effect of glucocorticoids when used as replacement therapy on bone remodelling in patients with adrenal insufficiency. Enhanced intracellular conversion of inactive cortisone to active cortisol, by 11 beta-hydroxysteroid dehydrogenase type 1(11β-HSD1) and other enzymes leading to alterations in glucocorticoid metabolism, may contribute to a deleterious effect on bone health in this patient group.

Animal data show that decreased activity of placental 11-beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which potently inactivates glucocorticoids (e.g. cortisol) to inert forms (cortisone), allows increased access of maternal glucocorticoids to the fetus and 'programs' hypertension. Data in humans are limited. We examined in humans the association between venous umbilical cord blood glucocorticoids, a potential marker for placental 11beta-HSD2 enzyme activity, and blood pressure at age 3 years.

The paraventricular hypothalamic nucleus (PVH) contains many neurons that innervate the brainstem, but information regarding their target sites remains incomplete. Here we labeled neurons in the rat PVH with an anterograde axonal tracer, Phaseolus vulgaris leucoagglutinin (PHAL), and studied their descending projections in reference to specific neuronal subpopulations throughout the brainstem. While many of their target sites were identified previously, numerous new observations were made. Major findings include: 1) In the midbrain, the PVH projects lightly to the ventral tegmental area, Edinger-Westphal nucleus, ventrolateral periaqueductal gray matter, reticular formation, pedunculopontine tegmental nucleus, and dorsal raphe nucleus. 2) In the dorsal pons, the PVH projects heavily to the pre-locus coeruleus, yet very little to the catecholamine neurons in the locus coeruleus, and selectively targets the viscerosensory subregions of the parabrachial nucleus. 3) In the ventral medulla, the superior salivatory nucleus, retrotrapezoid nucleus, compact and external formations of the nucleus ambiguous, A1 and caudal C1 catecholamine neurons, and caudal pressor area receive dense axonal projections, generally exceeding the PVH projection to the rostral C1 region. 4) The medial nucleus of the solitary tract (including A2 noradrenergic and aldosterone-sensitive neurons) receives the most extensive projections of the PVH, substantially more than the dorsal vagal nucleus or area postrema. Our findings suggest that the PVH may modulate a range of homeostatic functions, including cerebral and ocular blood flow, corneal and nasal hydration, ingestive behavior, sodium intake, and glucose metabolism, as well as cardiovascular, gastrointestinal, and respiratory activities.

The nucleus of the solitary tract (NTS) contains a unique subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). These neurons are mineralocorticoid-sensitive and are activated in association with salt appetite during sodium deficiency. In the absence of sodium deficiency, the HSD2 neurons and sodium appetite are both stimulated by chronic mineralocorticoid administration. After 7 days of treatment with deoxycorticosterone (2 mg/day), an increased number of HSD2 neurons became immunoreactive for the neuronal activity marker c-Fos. When given access to concentrated saline (3% NaCl), deoxycorticosterone-treated rats drank eight times more than vehicle-treated rats. Saline ingestion increased neuronal activation within the medial subdivision of the NTS, but the number of c-Fos-immunoreactive HSD2 neurons was reduced. This finding suggests that the HSD2 neurons are inhibited by signals directly related to saline ingestion, and not simply by the alleviation of sodium deficiency, which does not occur during mineralocorticoid administration.