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Objectives Gonococcal infections with a higher bacterial load may pose a higher risk of transmission. We assessed the association between gonococcal bacterial load and coinfection with Mycoplasma genitalium. Methods From September 2015 until May 2018, 200 men and transgender women who have sex with men participated in an HIV pre-exposure prophylaxis (PrEP) demonstration trial in Antwerp, Belgium. They underwent 3-monthly 3-site (anus, urine, and pharynx) molecular testing for N. gonorrhoeae and C. trachomatis and M. genitalium, irrespective of symptoms. Gonococcal bacterial load was determined on remnant DNA extracts using an in-house quantitative PCR. Results were expressed as log10 transformed copies/mL and analyzed with a linear regression model. Results Gonococcal bacterial load could be determined for 82 (80.4%) of 102 anal, 17 (73.9%) of 23 urine, and 64 (90.1%) of 71 pharyngeal samples. M. genitalium was detected in five of these anal, two urine, and two pharyngeal samples and C. trachomatis was detected in 16 anal, one urine, and two pharyngeal samples. Gonococcal bacterial load was significantly higher in the presence of M. genitalium (difference 0.92 log copies/mL, 95% CI 0.16–1.67). Conclusions Gonococcal bacterial load was higher with M. genitalium coinfection. M. genitalium may thus be a cofactor in gonococcal transmission.

Oestrogen and progesterone play essential roles in the release of mature oocytes, the priming and cycling of the uterine lining, and the maintenance of mammalian pregnancy. Progesterone is synthesizedde novoat the embryo implantation site in the mouse, during decidualization of the endometrium. During early stages of pregnancy, the locally produced progesterone is thought to act as an immunosuppressant, preventing rejection of the fetal allograft at the fetal-maternal interface. However, both uterine natural killer cells and dendritic cells express glucocorticoid receptor rather than progesterone receptor. The importance of glucocorticoids in early pregnancy is inferred from the presence of steroid receptors and the 11β-hydroxysteroid dehydrogenase enzymes, which modulate corticosterone action in the decidua, the trophoblast, the placenta, and the fetus. 11β-hydroxylase is the last enzyme in the metabolism of cholesterol to corticosterone and, in a mouse model of 11β-hydroxylase deficiency, complications of reproduction suggested its requirement for normal ovulation and uterine cell turnover. We present evidence that, in this model, folliculogenesis occurs normally but ovulation is inhibited, and abnormal uterine cell turnover ultimately leads to adenomyosis. Ovaries respond to a superovulation protocol by releasing oocytes and forming corpora lutea, and homozygous null blastocysts are capable of implantation, but the pregnancy is not maintained. We show that glucocorticoid is produced locally at the implantation site in control animals, revealing wide involvement of glucocorticoids in reproductive biology.

ObjectivesAntimicrobial resistance is generally linked to antimicrobial selection pressure. Antimicrobial-resistant Neisseria gonorrhoeae infections frequently emerge in core groups. We hypothesised that these groups are more often exposed to antimicrobials as a consequence of the repeated treatment of both symptomatic and asymptomatic sexually transmitted infections (STIs) and that frequent STI screening in asymptomatic patients may contribute indirectly to antimicrobial exposure. In this study, we explored the ecological association between screening intensity in men who have sex with men and antimicrobial susceptibility in N. gonorrhoeae in the USA.MethodsData on STI screening intensity came from the American Men’s Internet Survey between October 2014 and March 2015. Data on gonococcal susceptibility to azithromycin, ceftriaxone and cefixime were used from the Gonococcal Isolate Surveillance Project in 2015. Spearman’s correlation was used to determine the association between these two variables.ResultsA positive ecological association was found between STI screening intensity and geometric mean gonococcal minimum inhibitory concentration for ceftriaxone (rho=0.42, p=0.031) and cefixime (rho=0.42, p=0.029), but not for azithromycin (rho=0.31, p=0.11). The above results must be interpreted with caution as many limitations apply.ConclusionsVariation in STI screening intensity may contribute to differences in gonococcal resistance between States in the USA.

Antibiotic-sparing treatments are required to prevent the further emergence of antimicrobial resistance in Neisseria gonorrhoeae. Commensal Neisseria species have previously been found to inhibit the growth of pathogenic Neisseria species. For example, a previous study found that three out of five historical isolates of Neisseria mucosa could inhibit the growth of N. gonorrhoeae. In this study, we used agar overlay assays to assess if 24 circulating and historical isolates of Neisseria mucosa could inhibit the growth of 28 circulating and historical isolates of N. gonorrhoeae. Although pitting around each colony of N. mucosa created an optical illusion of decreased growth of N. gonorrhoeae, we found no evidence of inhibition (n = 24). In contrast, positive controls of Streptococcus pneumoniae and Escherichia coli demonstrated a strong inhibitory effect against the growth of N. gonorrhoeae.

We used agar overlay assays to assess if 24 circulating and historical isolates of Neisseria mucosa could inhibit the growth of 28 circulating and historical isolates of N. gonorrhoeae. We found no evidence of inhibition by N. mucosa (n=24). Positive controls Streptococcus pneumoniae and Escherichia coli demonstrated a strong inhibitory effect against the growth of N. gonorrhoeae.

BACKGROUND Men who have sex with men (MSM) experiencing recurrent sexually transmitted infections (STIs) may play a crucial role in the STI epidemic. However, there is limited understanding of what kind of behavior leads to recurrent STIs. METHODS A total of 179 MSM using preexposure prophylaxis were followed up for 18 months and were screened quarterly for chlamydia, gonorrhea, and syphilis from 2015 to 2018 in Belgium. Participants were stratified into 3 different groups (no STI, one STI episode, recurrent STI episodes during the study). Sociodemographic and sexual behavioral characteristics were compared between the 3 groups, and significant associations with recurrent STI were explored using multivariate logistic regression models. RESULTS A total of 62.0% (n = 111/179) of participants experienced at least one STI during the study, and more than 1 in 3 became reinfected with an STI at another visit (n = 66/179 [36.9%]). Participants experiencing recurrent STIs reported the highest frequency of sexualized drug use (86.4%) compared with participants experiencing one (60.0%) or no STI (47.1%). Therefore, sexualized drug use was highly associated with recurrent STIs (adjusted odds ratio [aOR]. 4.35). Other factors associated with recurrent STIs were being younger than 40 years (aOR, 3.29), had a high number (>4) of nonsteady partners with whom receptive (aOR, 1.17) or insertive (aOR, 1.12) condomless anal intercourse occurred in the last 3 months. CONCLUSIONS Sexualized drug use was the greatest risk factor for having recurrent STIs. Tailoring prevention and care, including specialized services tackling problematic drug use in a sexual context, may help to curb the STI epidemic among MSM.

Renal pericytes have a critical importance for angiogenesis and vascular remodeling, medullary blood flow regulation, and development of fibrosis. An emerging role for kidney pericytes is their ability to induce renin expression and synthesis. Here, we present methods for purification of human renal pericytes, their primary culture, and differentiation into renin-producing cells. Possible applications of these protocols include investigations into (1) renin cell recruitment mechanisms, (2) modulation of renin expression/secretion by small molecules, and (3) renin expression/secretion in nonrenal pericytes. A potential therapeutic application of this work is the identification of new players regulating the renin-angiotensin system.

The discovery of genetic mechanisms for resistance to obesity and diabetes may illuminate new therapeutic strategies for the treatment of this global health challenge. We used the polygenic 'lean' mouse model, which has been selected for low adiposity over 60 generations, to identify mitochondrial thiosulfate sulfurtransferase (Tst; also known as rhodanese) as a candidate obesity-resistance gene with selectively increased expression in adipocytes. Elevated adipose Tst expression correlated with indices of metabolic health across diverse mouse strains. Transgenic overexpression of Tst in adipocytes protected mice from diet-induced obesity and insulin-resistant diabetes. Tst-deficient mice showed markedly exacerbated diabetes, whereas pharmacological activation of TST ameliorated diabetes in mice. Mechanistically, TST selectively augmented mitochondrial function combined with degradation of reactive oxygen species and sulfide. In humans, TST mRNA expression in adipose tissue correlated positively with insulin sensitivity in adipose tissue and negatively with fat mass. Thus, the genetic identification of Tst as a beneficial regulator of adipocyte mitochondrial function may have therapeutic significance for individuals with type 2 diabetes N. Morton was supported by a Career Development Fellowship, Institutional Strategic Support Fund award and a New Investigator Award from the Wellcome Trust (100981/Z/13/Z), a Research Councils UK Fellowship and a British Heart Foundation Centre of Research Excellence exchange award. We thank the Slovenian Research Agency (core funding P4-0220, project N5-0003 Syntol and J4-6804 to S.H.); and a Young Scientist Fellowship to J. Beltram. We acknowledge support of the British Heart Foundation Research Excellence Award in support of the Bioinformatics Core contribution. T. Stulnig received funding from the Federal Ministry of Economy, Family and Youth and the Austrian National Foundation for Research, Technology and Development. G. Churchill was supported by the US National Institutes of Health grant R01GM 070683. J.M. Fernandez-Real acknowledges funding from FIS PI11/00214. A. Vidal-Puig was funded by the UK Medical Research Council (MRC) MDU, an MRC Programme grant, MRC DMC Core and MITIN (HEALTH-F4-2008-223450)

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P Scarb1, Sqle, Apoa1) and disposal (Cyp27a1, Cyp7b1). Oil red O staining showed lipid depletion consistent with reduced expression of genes involved in lipid synthesis. Genes involved in steroidogenesis (Star, Cyp11a1, Cyp11b1) were modestly affected (P Ki67, Ccna2, Ccnb2 and Tk1 expression complemented immunohistochemical evidence of a 3-fold change in cell proliferation. Growth arrest genes, Cdkn1a and Cdkn1c, which are known to be active in hypertrophied cells, were increased >4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu,) were downregulated and apoptotic cells (Tunel staining) were fewer (P

Background The direct detection of Treponema pallidum peptides in bodily fluids could facilitate the early diagnosis of initial-, repeat-, congenital- and neuro-syphilis. Methods To this end, we prospectively recruited 54 individuals with a new diagnosis of syphilis and 6 controls. Their urine specimens were pooled according to disease stage and assessed using complementary mass spectrometry techniques (MALDI-TOF-TOFMS/MS, LC/ESI-IM-Q-TOF/HDMSE) to uncover potential syphilis biomarkers. Results In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins (Tp0486, Tp0742, Tp0804 and Tp0369) that have no, or minimal, genetic sequence similarity to other known proteins, including prokaryotes and human proteins. Conclusion This is the first study reporting direct detection of T. pallidum proteins in human biofluid samples using MS-based proteomics methods. These could be promising diagnostic test targets. Disclosure No significant relationships.

Background Previous transformation experiments with Neisseria gonorrhoeae (Ng) have established that it is able to acquire high-level antibiotic resistance via transformation. We aimed to assess if a high-level ceftriaxone resistant Ng strain (WHO X) was able to acquire resistance to azithromycin (AZM) via this mechanism. Methods A mid log phase culture of Ng WHO X (AZM MIC 0.25 µg/mL) was mixed with whole genomic DNA extracted from Ng WHO strain V (AZM ≥ 256 µg/mL). A concentration of 1,5x MIC of AZM was added as a stress factor for the selection of the resistance determinants. Control experiments were conducted by omitting the addition of AZM and/or DNA. Consecutively, the mixture was plated on blood agar plates and incubated at 36°C in a 6% CO2 atmosphere. Of each blood agar plate 1 or 2 colonies were selected for E testing performed according to CLSI guidelines. Colonies growing alongside the E-test strip at the higher range of the MIC values were selected for further characterization, including whole genome sequencing, and to identify the acquired resistance mechanisms. Results The MIC for AZM of WHO strain X increased to greater than 256 μg/ml. There was no change in the MICs of Ng in the control experiments. Whole genome sequencing results will be presented demonstrating the pathways to resistance. Conclusion Neisseria gonorrhoeae is able to rapidly acquire high level macrolide resistance in the presence of both DNA of AZM highly resistant NG strains and AZM. Disclosure No significant relationships.

Background Current serology-based, treponemal-specific diagnostic tests detect antibodies reactive against T. pallidum molecules and cannot differentiate between past and current syphilis infections. Further, existing diagnostic tests for syphilis have non-optimal sensitivity and specificity and require expertise for test administration and interpretation. These limitations, combined with the rising number of syphilis infections, highlight the need for a reliable direct diagnostic assay to detect active syphilis. We sought to develop such an assay using immuno-mass spectrometry to detect T. pallidum proteins. Our results revealed that select T. pallidum proteins are glycosylated. Methods We developed antibodies directed against proteotypic, surrogate peptides from six prioritized T. pallidum biomarker proteins. These antibodies were tested using a technology called Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA), which involves antibody enrichment of the peptide surrogates coupled with their identification by mass spectrometry. The anti-peptide antibodies were tested by SISCAPA and in immunoblots to detect T. pallidum proteins in urine from patients with clinically confirmed active syphilis. Results Immunoblot analyses consistently identified T. pallidum proteins in urine samples from patients with syphilis. Initially, SISCAPA technology detected the surrogate peptides in only a fraction of the urine samples. However, deglycosylation of the proteins in the urine samples prior to SISCAPA analyses allowed successful identification of the T. pallidum biomarkers. Conclusion This is the first report of protein glycosylation during T. pallidum infection. The results show sample deglycosylation prior to SISCAPA analysis improves peptide detection and enables use of a SISCAPA-based direct diagnostic test for accurately detecting active syphilis. Further, the results suggest a potential mechanism of immune evasion used during infection, that of masking T. pallidum proteins from the immune system by the addition of glycosyl groups. These findings increase our understanding of T. pallidum infection and will assist with development of a non-invasive, sensitive and specific assay for syphilis. Disclosure No significant relationships.

Background The in-vitro genetic pathways to high-level azithromycin (AZM) resistance have hitherto not been established in Neisseria gonorrhoeae (Ng). Methods A Ng morbidostat that dynamically increases AZM concentrations in response to Ng growth was built according to the protocol of Toprak et al. The reference strains Ng WHO-F and WHO-X were grown in 12 mL GC broth supplemented with IsoVitaleX™ (1%) and vancomycin, colistin, nystatin, trimethoprim selective supplement for 30 days in a 6% CO2 environment at 36°C. Depending on the turbidity and growth of the culture, 1 mL of fresh medium or AZM was added to the culture vials after each cycle of 21 minutes. The experiment started with a concentration of 20x minimal inhibitory concentration (MIC) of AZM in the drug reservoir which was increased up to 320x MIC for both strains by the end of the experiment. Samples of the cultures were taken 2–3 times a week and MICs of AZM were determined using E-tests. Whole genome sequencing will be performed using Illumina MiSeq. All experiments were run in triplicate. Results The initial MICs of WHO-F and WHO-X were 0,125 µg/mL and 0,25 µg/mL respectively. In the first week, the MICs of WHO-F and WHO-X increased approximately 24-fold for WHO-F and 48-fold for WHO-X. After 30 days, WHO-F and WHO-X had attained MICs of 96 µg/mL and ≥296 µg/mL, respectively. The genetic pathways to resistance will be analysed and presented. Conclusion We were able to induce high level AZM resistance in Ng within 30 days of AZM exposure using our Ng morbidostat. Disclosure No significant relationships.

Mechanisms to explain post-prandial increases in circulating glucocorticoids are not well understood and may involve increased adrenal secretion and/or altered steroid metabolism. We have compared salivary levels of cortisol and cortisone levels in healthy male and female volunteers fed either a low or cholesterol-rich midday meal. Urinary levels of steroids, bile acids and markers of lipid peroxidation were also measured. Males and females showed expected circadian changes in salivary steroids and postprandial peaks within 1h of feeding. After a high-cholesterol meal, postprandial cortisol increases were higher in males whereas post-prandial cortisone levels were higher in females. Urinary cortisol but not cortisone levels were higher on the day when males and females ate a high-cholesterol meal. Urinary bile acid excretion and anti-oxidant markers of lipid peroxidation, thiobarbituric acid reactive substances (TBARS), and total phenol content were not affected by dietary cholesterol but tended to be higher in males. Cross-tabulation of correlation coefficients indicated positive associations between urinary markers of peroxidation, bile acids, and cortisol:cortisone ratios. We conclude that dietary cholesterol (a substrate for steroidogenesis) does not have an acute effect on adrenal glucocorticoid synthesis and that gender but not a high-cholesterol meal may influence the interconversion of cortisol and cortisone. Longer term studies of the effects of dietary cholesterol are needed to analyze the associations between bile acids, steroid metabolism, and secretion and lipid peroxidation.

Malaria reduces host fitness and survival by pathogen-mediated damage and inflammation. Disease tolerance mechanisms counter these negative effects without decreasing pathogen load. Here, we demonstrate that in four different mouse models of malaria, adrenal hormones confer disease tolerance and protect against early death, independently of parasitemia. Surprisingly, adrenalectomy differentially affects malaria-induced inflammation by increasing circulating cytokines and inflammation in the brain but not in the liver or lung. Furthermore, without affecting the transcription of hepatic gluconeogenic enzymes, adrenalectomy causes exhaustion of hepatic glycogen and insulin-independent lethal hypoglycemia upon infection. This hypoglycemia is not prevented by glucose administration or TNF-α neutralization. In contrast, treatment with a synthetic glucocorticoid (dexamethasone) prevents the hypoglycemia, lowers cerebral cytokine expression and increases survival rates. Overall, we conclude that in malaria, adrenal hormones do not protect against lung and liver inflammation. Instead, they prevent excessive systemic and brain inflammation and severe hypoglycemia, thereby contributing to tolerance., Disease tolerance mechanisms counter the negative effects of infection without decreasing the pathogen load. Here, the authors show that in mouse models of malaria, such disease tolerance can be conferred by adrenal hormones, by preventing excessive inflammation and hypoglycemia.

New Findings What is the central question of this study? Glucocorticoids act in the kidney to promote salt and water retention. Renal 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), by increasing local concentrations of glucocorticoids, may exert an antinatriuretic effect. We hypothesized that global deletion of 11βHSD1 in the mouse would give rise to a salt-wasting renal phenotype. What is the main finding and its importance? We subjected a mouse model of global 11βHSD1 deletion to studies of water and electrolyte balance, renal clearance, urinary steroid excretion, renin–angiotensin system activation and renal sodium transporter expression. We found no significant effects on renal sodium or water excretion. Any effect of renal 11βHSD1 on sodium homeostasis is subtle. Glucocorticoids act in the kidney to regulate glomerular haemodynamics and tubular sodium transport; the net effect favours sodium retention. 11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) is expressed in the renal tubules and the interstitial cells of the medulla, where it is likely to regenerate active glucocorticoids from inert 11-keto forms. The physiological function of renal 11βHSD1 is largely unknown. We hypothesized that loss of renal 11βHSD1 would result in salt wasting and tested this in a knockout mouse model in which 11βHSD1 was deleted in all body tissues. In balance studies, 11βHSD1 deletion had no effect on water, sodium or potassium metabolism; transition to a low-sodium diet did not reveal a natriuretic phenotype. Renal clearance studies demonstrated identical haemodynamic parameters (arterial blood pressure, renal blood flow and glomerular filtration rate) in knockout and wild-type mice, but revealed an augmented kaliuretic response to thiazides in 11βHSD1 knockout animals. There was no effect on the natriuretic response to the amiloride analogue benzamil. Urinary excretion of deoxycorticosterone was higher in 11βHSD1 knockout mice, and there was hypertrophy of cells in the zona fasciculata of the adrenal cortex. There was no difference in the activity of the renin–angiotensin and nitric oxide systems, no difference in renal histology and no difference in the abundance of key tubular transporter proteins. We conclude that any effect of 11βHSD1 on renal sodium excretion is subtle.

Obesity and hypertension are 2 major health issues of the 21st century. The syndrome of apparent mineralocorticoid excess is caused by deficiency of 11β-hydroxysteroid dehydrogenase type 2 (Hsd11b2), which normally inactivates glucocorticoids, rendering the mineralocorticoid receptor aldosterone-specific. The metabolic consequences of Hsd11b2 knockout in the rat are investigated in parallel with electrolyte homeostasis. Hsd11b2 was knocked out, by pronuclear microinjection of targeted zinc-finger nuclease mRNAs, and 1 line was characterized for its response to renal and metabolic challenges. Plasma 11-dehydrocorticosterone was below detection thresholds, and Hsd11b2 protein was undetected by Western blot, indicating complete ablation. Homozygotes were 13% smaller than wild-type littermates, and were polydipsic and polyuric. Their kidneys, adrenals, and hearts were significantly enlarged, but mesenteric fat pads and liver were significantly smaller. On a 0.3% Na diet, mean arterial blood pressure was ≈65 mm Hg higher than controls but only 25 mm Hg higher on a 0.03% Na(+) diet. Urinary Na/K ratio of homozygotes was similar to controls on 0.3% Na(+) diet but urinary albumin and calcium were elevated. Corticosterone and aldosterone levels showed normal circadian variation on both a 0.3% and 0.03% Na(+) diet, but plasma renin was suppressed in homozygotes on both diets. Plasma glucose responses to an oral glucose challenge were reduced despite low circulating insulin, indicating much greater sensitivity to insulin in homozygotes. The rat model reveals mechanisms linking electrolyte homeostasis and metabolic control through the restriction of Hsd11b1 substrate availability.

Na(+) transport in the renal distal convoluted tubule (DCT) by the thiazide-sensitive NaCl cotransporter (NCC) is a major determinant of total body Na(+) and BP. NCC-mediated transport is stimulated by aldosterone, the dominant regulator of chronic Na(+) homeostasis, but the mechanism is controversial. Transport may also be affected by epithelial remodeling, which occurs in the DCT in response to chronic perturbations in electrolyte homeostasis. Hsd11b2(-/-) mice, which lack the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) and thus exhibit the syndrome of apparent mineralocorticoid excess, provided an ideal model in which to investigate the potential for DCT hypertrophy to contribute to Na(+) retention in a hypertensive condition. The DCTs of Hsd11b2(-/-) mice exhibited hypertrophy and hyperplasia and the kidneys expressed higher levels of total and phosphorylated NCC compared with those of wild-type mice. However, the striking structural and molecular phenotypes were not associated with an increase in the natriuretic effect of thiazide. In wild-type mice, Hsd11b2 mRNA was detected in some tubule segments expressing Slc12a3, but 11βHSD2 and NCC did not colocalize at the protein level. Thus, the phosphorylation status of NCC may not necessarily equate to its activity in vivo, and the structural remodeling of the DCT in the knockout mouse may not be a direct consequence of aberrant corticosteroid signaling in DCT cells. These observations suggest that the conventional concept of mineralocorticoid signaling in the DCT should be revised to recognize the complexity of NCC regulation by corticosteroids.

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20β-dihydrocortisol (20β-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20β-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20β-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.

Highlights • Spatial memory deficits after midlife stress remain for 6 months. • Enhanced fear memory and impaired extinction 6 months after midlife stress. • Increased anxiety and depressive-like behaviors 7 months after midlife stress. • 11β-HSD1 deficient mice resist enduring effects of midlife stress on behaviors., Chronic exposure to stress during midlife associates with subsequent age-related cognitive decline and may increase the vulnerability to develop psychiatric conditions. Increased hypothalamic-pituitary-adrenal (HPA) axis activity has been implicated in pathogenesis though any causative role for glucocorticoids is unestablished. This study investigated the contribution of local glucocorticoid regeneration by the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), in persisting midlife stress-induced behavioral effects in mice. Middle-aged (10 months old) 11β-HSD1-deficient mice and wild-type congenic controls were randomly assigned to 28 days of chronic unpredictable stress or left undisturbed (non-stressed). All mice underwent behavioral testing at the end of the stress/non-stress period and again 6–7 months later. Chronic stress impaired spatial memory in middle-aged wild-type mice. The effects, involving a wide spectrum of behavioral modalities, persisted for 6–7 months after cessation of stress into early senescence. Enduring effects after midlife stress included impaired spatial memory, enhanced contextual fear memory, impaired fear extinction, heightened anxiety, depressive-like behavior, as well as reduced hippocampal glucocorticoid receptor mRNA expression. In contrast, 11β-HSD1 deficient mice resisted both immediate and enduring effects of chronic stress, despite similar stress-induced increases in systemic glucocorticoid activity during midlife stress. In conclusion, chronic stress in midlife exerts persisting effects leading to cognitive and affective dysfunction in old age via mechanisms that depend, at least in part, on brain glucocorticoids generated locally by 11β-HSD1. This finding supports selective 11β-HSD1 inhibition as a novel therapeutic target to ameliorate the long-term consequences of stress-related psychiatric disorders in midlife.

We have previously demonstrated that neutrophil recruitment to the heart following myocardial infarction (MI) is enhanced in mice lacking 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) that regenerates active glucocorticoid within cells from intrinsically inert metabolites. The present study aimed to identify the mechanism of regulation. In a mouse model of MI, neutrophil mobilization to blood and recruitment to the heart were higher in 11β-HSD1-deficient (Hsd11b1−/−) relative to wild-type (WT) mice, despite similar initial injury and circulating glucocorticoid. In bone marrow chimeric mice, neutrophil mobilization was increased when 11β-HSD1 was absent from host cells, but not when absent from donor bone marrow-derived cells. Consistent with a role for 11β-HSD1 in ‘host’ myocardium, gene expression of a subset of neutrophil chemoattractants, including the chemokines Cxcl2 and Cxcl5, was selectively increased in the myocardium of Hsd11b1−/− mice relative to WT. SM22α-Cre directed disruption of Hsd11b1 in smooth muscle and cardiomyocytes had no effect on neutrophil recruitment. Expression of Cxcl2 and Cxcl5 was elevated in fibroblast fractions isolated from hearts of Hsd11b1−/− mice post MI and provision of either corticosterone or of the 11β-HSD1 substrate, 11-dehydrocorticosterone, to cultured murine cardiac fibroblasts suppressed IL-1α-induced expression of Cxcl2 and Cxcl5. These data identify suppression of CXCL2 and CXCL5 chemoattractant expression by 11β-HSD1 as a novel mechanism with potential for regulation of neutrophil recruitment to the injured myocardium, and cardiac fibroblasts as a key site for intracellular glucocorticoid regeneration during acute inflammation following myocardial injury.

While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11β-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research.

In aldosterone target tissues, 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is coexpressed with mineralocorticoid receptors (MR) and protects the receptor from activation by glucocorticoids. Null mutations in the encoding gene, HSD11B2, cause apparent mineralocorticoid excess, in which hypertension is thought to reflect volume expansion secondary to sodium retention. Hsd11b2−/−mice are indeed hypertensive, but impaired natriuretic capacity is associated with significant volume contraction, suggestive of a urine concentrating defect. Water turnover and the urine concentrating response to a 24-h water deprivation challenge were therefore assessed in Hsd11b2−/−mice and controls. Hsd11b2−/−mice have a severe and progressive polyuric/polydipsic phenotype. In younger mice (∼2 mo of age), polyuria was associated with decreased abundance of aqp2 and aqp3 mRNA. The expression of other genes involved in water transport ( aqp4, slc14a2, and slc12a2) was not changed. The kidney was structurally normal, and the concentrating response to water deprivation was intact. In older Hsd11b2−/−mice (>6 mo), polyuria was associated with a severe atrophy of the renal medulla and downregulation of aqp2, aqp3, aqp4, slc14a2, and slc12a2. The concentrating response to water deprivation was impaired, and the natriuretic effect of the loop diuretic bumetanide was lost. In older Hsd11b2−/−mice, the V2 receptor agonist desmopressin did not restore full urine concentrating capacity. We find that Hsd11b2−/−mice develop nephrogenic diabetes insipidus. Gross changes to renal structure are observed, but these were probably secondary to sustained polyuria, rather than of developmental origin.

BACKGROUND AND PURPOSE Dissociating anti-inflammatory efficacy from the metabolic side effects of glucocorticoids is an attractive therapeutic goal. 5α-Tetrahydro-corticosterone (5αTHB), produced from corticosterone by 5α-reductases, activates glucocorticoid receptors. This study compares the effects of 5αTHB on inflammation and metabolism in vitro and in vivo. METHODS Suppression of cytokine release by 5αTHB and corticosterone were studied following LPS activation of mouse bone marrow derived macrophages. In vivo the efficacy of these steroids to dysregulate metabolic homeostasis and modulate immune suppression and the responses to thioglycollate-induced peritonitis in C57BL/6 mice were studied following acute injection (1.5–15 mg) and chronic infusion (50 µg·day−1, 14 days). RESULTS In macrophages, 5αTHB increased secretion of IL-10 similarly to corticosterone (180%, 340%; data are % vehicle, treated with 5αTHB and corticosterone, respectively) and suppressed LPS-induced secretion of TNF-α (21.9%, 74.2%) and IL-6 (16.4%, 69.4%). In mice with thioglycollate-induced peritonitis, both 5αTHB and corticosterone reduced the numbers of neutrophils (58.6%, 49.9%) and inflammatory monocytes (69.5%, 96.4%), and also suppressed MCP-1 (48.7%, 80.9%) and IL-6 (53.5%, 86.7%) in peritoneal exudate. In mice chronically infused with 5αTHB and corticosterone LPS-induced production of TNF-α from whole blood was suppressed to the same degree (63.2%, 37.2%). However, in contrast to corticosterone, 5αTHB did not induce body weight loss, increase blood pressure or induce hyperinsulinaemia. CONCLUSIONS 5αTHB has anti-inflammatory effects in vitro and in vivo. At doses with equivalent anti-inflammatory efficacy to corticosterone, 5αTHB did not induce metabolic toxicity and thus may be a prototype for a safer anti-inflammatory drug.

Salt sensitivity of blood pressure is an independent risk factor for cardiovascular morbidity. Mechanistically, abnormal mineralocorticoid action and subclinical renal impairment may blunt the natriuretic response to high sodium intake, causing blood pressure to rise. 11β-Hydroxysteroid dehydrogenase type 2 (11βHSD2) controls ligand access to the mineralocorticoid receptor, and ablation of the enzyme causes severe hypertension. Polymorphisms in HSD11B2 are associated with salt sensitivity of blood pressure in normotensives. In this study, we used mice heterozygote for a null mutation in Hsd11b2 ( Hsd11b2 +/− ) to define the mechanisms linking reduced enzyme activity to salt sensitivity of blood pressure. A high-sodium diet caused a rapid and sustained increase in blood pressure in Hsd11b2 +/− mice but not in wild-type littermates. During the adaptation to high-sodium diet, heterozygotes displayed impaired sodium excretion, a transient positive sodium balance, and hypokalemia. After 21 days of high-sodium feeding, Hsd11b2 +/− mice had an increased heart weight. Mineralocorticoid receptor antagonism partially prevented the increase in heart weight but not the increase in blood pressure. Glucocorticoid receptor antagonism prevented the rise in blood pressure. In Hsd11b2 +/− mice, high-sodium feeding caused suppression of aldosterone and a moderate but sustained increase in corticosterone. This study demonstrates an inverse relationship among 11βHSD2 activity, heart weight, and blood pressure in a clinically important context. Reduced activity causes salt sensitivity of blood pressure, but this does not reflect illicit activation of mineralocorticoid receptors by glucocorticoids. Instead, we have identified a novel interaction among 11βHSD2, dietary salt, and circulating glucocorticoids.

Nat. Med. 22, 771–779 (2016); published online 6 June 2016; corrected after print 7 March 2018 In the version of this article initially published, the colors of the lines were switched in the graph that shows the glucose infusion rates for wild-type mice and Adipoq-Tst transgenic mice in Figure 3b. The top line should be purple, and the bottom line should be black.

We investigated the effects on urinary steroid and electrolyte excretion and renal gene expression of chronic infusions of ACTH in the mouse. ACTH caused a sustained increase in corticosteroid excretion; aldosterone excretion was only transiently elevated. There was an increase in the excretion of deoxycorticosterone, a weak mineralocorticoid, to levels of physiological significance. Nevertheless, we observed neither antinatriuresis nor kaliuresis in ACTH-treated mice, and plasma renin activity was not suppressed. We identified no changes in expression of mineralocorticoid target genes. Water turnover was increased in chronic ACTH-treated mice, as were hematocrit and hypertonicity: volume contraction is consistent with high levels of glucocorticoid. ACTH-treated mice exhibited other signs of glucocorticoid excess, such as enhanced weight gain and involution of the thymus. We identified novel ACTH-induced changes in 1) genes involved in vitamin D ( Cyp27b1, Cyp24a1, Gc) and calcium ( Sgk, Calb1, Trpv5) metabolism associated with calciuria and phosphaturia; 2) genes that would be predicted to desensitize the kidney to glucocorticoid action ( Nr3c1, Hsd11b1, Fkbp5); and 3) genes encoding transporters of enzyme systems associated with xenobiotic metabolism and oxidative stress. Although there is evidence that ACTH-induced hypertension is a function of physiological cross talk between glucocorticoids and mineralocorticoids, the present study suggests that the major changes in electrolyte and fluid homeostasis and renal function are attributable to glucocorticoids. The calcium and organic anion metabolism pathways that are affected by ACTH may explain some of the known adverse effects associated with glucocorticoid excess.

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11β-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone = 0.735% and corticosterone = 0.045%), and for B assay (aldosterone = 0.14%, 11-dehydro-B = 0.006%, cortisol = 0.016% and DOC = 0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y = 1.092X − 0.054, R2 = 0.988; B ELISA: Y = 1.047X − 0.226, R2 = 0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.

Background Clinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases. Methods Antibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones. Results Cross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R2 = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice. Conclusion We describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia.

Patients with congenital adrenal hyperplasia arising from mutations of 11beta-hydroxylase, the final enzyme in the glucocorticoid biosynthetic pathway, exhibit glucocorticoid deficiency, adrenal hyperplasia driven by unsuppressed hypothalamo-pituitary-adrenal activity, and excess mineralocorticoid activity caused by the accumulation of deoxycorticosterone. A mouse model, in which exons 3-7 of Cyp11b1 (the gene encoding 11beta-hydroxylase) were replaced with cDNA encoding enhanced cyan fluorescent protein, was generated to investigate the underlying disease mechanisms. Enhanced cyan fluorescent protein was expressed appropriately in the zona fasciculata of the adrenal gland, and targeted knock-out was confirmed by urinary steroid profiles and, immunocytochemically, by the absence of 11beta-hydroxylase. The null mice exhibited glucocorticoid deficiency, mineralocorticoid excess, adrenal hyperplasia, mild hypertension, and hypokalemia. They also displayed glucose intolerance. Because rodents do not synthesize adrenal androgens, changes in reproductive function such as genital virilization of females were not anticipated. However, adult homozygote females were infertile, their ovaries showing an absence of corpora lutea and a central proliferation of disorganized steroidogenic tissue. Null females responded normally to superovulation, suggesting that raised systemic progesterone levels also contribute to infertility problems. The model reveals previously unrecognized phenotypic subtleties of congenital adrenal hyperplasia.

We determined whether altered physical activity levels might underlie the contrasting adiposity of a divergently selected polygenic murine model of metabolic syndrome (Fat; F) and leanness (Lean; L) mice. We measured physical activity with a long term running wheel experiment and performed an additional high fat diet intervention. Further, we measured posture allocation by visual monitoring within the home cage as a non-exercise correlate of 'normal' physical activity. Whilst initially similar, running wheel activity of the F line declined with age, while the activity of the L line increased. Food intake was higher in the L line and increased with wheel exposure. Vertical rearing measured by video quantification in the home cage, without the stimulus of a running wheel was also significantly higher in the L line. The two lines developed novel alternate strategies to defend their body weight when exposed to high fat diets with a running wheel. F mice increased their running wheel activity, and despite unaltered food intake, still gained weight. L mice reduced their food intake and maintained activity levels without a significant change in body weight. Phenotypic selection for divergence in body fat content has co-segregated with a genetic predisposition for divergent physical activity levels and different strategies for coping with exposure to high fat diets that will facilitate the discovery of the genes underlying these important obesity related traits.

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 μg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either gene's expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels.

Low birth weight in humans is predictive of hypertension in adult life. Although the mechanisms underlying this link remain unknown, fetal overexposure to glucocorticoids has been implicated. We previously showed that prenatal dexamethasone (DEX) exposure in the rat lowers birth weight and programmes adult hypertension. The current study aimed to further investigate the nature of this hypertension and to elucidate its origins. Unlike previous studies, we assessed offspring blood pressure (BP) with radiotelemetry, which is unaffected by stress artefacts of measurement. We show that prenatal DEX during the last week of pregnancy results in offspring of low birth weight (14% reduction) that have lower basal BP in adulthood (similar to 4-8 mmHg lower); with the commonly expected hypertensive phenotype only being noted when these offspring are subjected to even mild disturbance or a more severe stressor (up to 30 mmHg higher than controls). Moreover, DEX-treated offispring sustain their stress-induced hypertension for longer. Promotion of systemic catecholamine release (amphetamine) induced a significantly greater rise of BP in the DEX animals (77% increase) over that observed in the vehicle controls. Additionally, we demonstrate that the isolated mesenteric vasculature of DEX-treated offspring display greater sensitivity to noradrenaline and other vasoconstrictors. We therefore conclude that altered sympathetic responses mediate the stress-induced hypertension associated with prenatal DEX programming.

The nuclear mineralocorticoid receptor (MR), a high-affinity receptor for glucocorticoids, is highly expressed in the hippocampus where it underpins cognitive, behavioural and neuroendocrine regulation. Increased neuronal MR expression occurs early in the response to cellular injury in vivo and in vitro and is associated with enhanced neuronal survival. To determine whether increased neuronal MR might be causal in protecting against ischaemic damage in vivo we generated a forebrain-specific MR-overexpressing transgenic mouse (MR-Tg) under the control of the CamKII alpha promoter, and subjected mice to transient cerebral global ischaemia induced by bilateral common carotid artery occlusion for 20 min. We also separately assessed the effects of MR overexpression on hypothalamic-pituitary-adrenal (HPA) axis activity and cognitive and affective functions in noninjured animals. Our results showed that MR-Tg mice had significantly reduced neuronal death following transient cerebral global ischaemia compared to wild-type littermates. This effect was not associated with alterations in basal or poststress HPA axis function or in arterial blood pressure. MR-Tg mice also demonstrated improved spatial memory retention, reduced anxiety and altered behavioural response to novelty. The induction of neuronal MR appears to offer a protective response which has potential therapeutic implications in cerebral ischaemia and cognitive and affective disorders.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) acts as a reductase in vivo, regenerating active glucocorticoids within cells from circulating inert 11-keto forms, thus amplifying local glucocorticoid action. 11beta-HSD1 is predominantly expressed in liver and also adipose tissue and brain. Mice deficient in 11beta-HSD1 (11beta-HSD1(-/-)) exhibit adrenal hyperplasia, raised basal corticosterone levels, and increased hypothalamic-pituitary-adrenal (HPA) axis responses to stress. Whereas reduced peripheral glucocorticoid regeneration may explain adrenal hypertrophy and exaggerated stress responses, elevated basal glucocorticoid levels support a role for 11beta-HSD1 within the brain in amplifying glucocorticoid feedback. To test this hypothesis, apolipoprotein E-HSD1 mice overexpressing 11beta-HSD1 in liver were intercrossed with 11beta-HSD1(-/-) mice to determine whether complementation of hepatic 11beta-HSD1 can restore adrenal and HPA defects. Transgene-mediated delivery of 11beta-HSD1 activity to the liver rescued adrenal hyperplasia and reversed exaggerated HPA stress responses in 11beta-HSD1(-/-) mice. Unexpectedly, elevated nadir plasma corticosterone levels were also restored to control levels. Consistent with this, CYP11B1 mRNA expression in the adrenal cortex of 11beta-HSD1(-/-) mice was increased by 50% but returned to control levels in 11beta-HSD1(-/-) mice bearing the apolipoprotein E-HSD1 transgene. 11beta-HSD1(-/-) mice have lower plasma glucose levels, but the fall in plasma corticosterone with sucrose supplementation was similar in 11beta-HSD1(-/-) and control mice, suggesting glucose deficiency is not the main mechanism whereby basal corticosterone levels are elevated in the null mice. Thus, regeneration of glucocorticoids by 11beta-HSD1 in the liver normalizes all aspects of HPA axis dysregulation in 11beta-HSD1(-/-) mice, without restoration of enzyme activity in key feedback areas of the forebrain. Therefore, hepatic glucocorticoid metabolism influences basal as well as stress-associated functions of the HPA axis.

What is the central question of this study? Glucocorticoids act in the kidney to promote salt and water retention. Renal 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), by increasing local concentrations of glucocorticoids, may exert an antinatriuretic effect. We hypothesized that global deletion of 11βHSD1 in the mouse would give rise to a salt-wasting renal phenotype. What is the main finding and its importance? We subjected a mouse model of global 11βHSD1 deletion to studies of water and electrolyte balance, renal clearance, urinary steroid excretion, renin-angiotensin system activation and renal sodium transporter expression. We found no significant effects on renal sodium or water excretion. Any effect of renal 11βHSD1 on sodium homeostasis is subtle. Glucocorticoids act in the kidney to regulate glomerular haemodynamics and tubular sodium transport; the net effect favours sodium retention. 11β-Hydroxysteroid dehydrogenase type 1 (11βHSD1) is expressed in the renal tubules and the interstitial cells of the medulla, where it is likely to regenerate active glucocorticoids from inert 11-keto forms. The physiological function of renal 11βHSD1 is largely unknown. We hypothesized that loss of renal 11βHSD1 would result in salt wasting and tested this in a knockout mouse model in which 11βHSD1 was deleted in all body tissues. In balance studies, 11βHSD1 deletion had no effect on water, sodium or potassium metabolism; transition to a low-sodium diet did not reveal a natriuretic phenotype. Renal clearance studies demonstrated identical haemodynamic parameters (arterial blood pressure, renal blood flow and glomerular filtration rate) in knockout and wild-type mice, but revealed an augmented kaliuretic response to thiazides in 11βHSD1 knockout animals. There was no effect on the natriuretic response to the amiloride analogue benzamil. Urinary excretion of deoxycorticosterone was higher in 11βHSD1 knockout mice, and there was hypertrophy of cells in the zona fasciculata of the adrenal cortex. There was no difference in the activity of the renin-angiotensin and nitric oxide systems, no difference in renal histology and no difference in the abundance of key tubular transporter proteins. We conclude that any effect of 11βHSD1 on renal sodium excretion is subtle.

Excessive exposure to glucocorticoids during gestation reduces birth weight and induces permanent hypertension in adulthood. The mechanisms underlying this programmed elevation of blood pressure have not been established. We hypothesised that prenatal glucocorticoid exposure may lead to vascular dysfunction in adulthood. Pregnant rats received dexamethasone (Dex) (100 μg/kg, s.c.) or vehicle (control) daily throughout pregnancy. Blood pressure was elevated (students t-test, unpaired; P < 0.05) in adult female offspring (aged 12–16 weeks) of Dex-treated mothers (148.0 ± 3.6 mmHg, n=10) compared with the control group (138.0 ± 2.5 mmHg, n=8). Vascular responsiveness in aortae and mesenteric arteries was differentially affected by prenatal Dex: aortae were less responsive to angiotensin II, whereas mesenteric arteries were more responsive to norepinephrine, vasopressin and potassium (mesenteric arteries respond poorly to angiotensin II in vitro). Acetylcholine-mediated, endothelium-dependent relaxation was similar in both groups. Prenatal exposure to Dex had no effect on blood pressure or aldosterone response to acute (15 min, i.v.) infusion of angiotensin II (75 ng/kg per min). In contrast, chronic (2-week, s.c.) infusion of angiotensin II (100 ng/kg per min) produced a greater elevation (P < 0.05) of blood pressure in Dex-treated rats (150.0 ± 3.6 mmHg) than in controls (135.3 ± 5.4 mmHg), and aldosterone levels were higher in Dex-treated animals. There was no angiotensin II-induced medial hypertrophy/hyperplasia in mesenteric arteries from Dex-treated rats. These results indicate that vascular function is altered in a region-specific manner in rats with glucocorticoid-programmed hypertension. Despite a striking increase in mesenteric artery contraction in Dex-treated rats, in vivo studies suggest that abnormalities of the renin-angiotensin-aldosterone system, rather than enhanced vascular contractility, may be responsible for the elevation of blood pressure in these animals.

Glucocorticoid overexposure in utero may underlie the association between low birth weight and subsequent development of common cardiovascular and metabolic pathologies. Previously, we have shown that prenatal dexamethasone (DEX) exposure in rat reduces birth weight and programs the hypothalamic-pituitary axis and fasting and postprandial hyperglycemia in adult males and hypertension in adult males and females. This study aimed to determine 1) whether there were gender differences in prenatal DEX-programmed offspring, and 2) whether the renin-angiotensin system (RAS) plays a role in the programming of hypertension. Rats exposed to DEX in utero (100 μg·kg−1·day−1 from embryonic days 14–21) were of lower birth weight (by 12%, P < 0.01) and displayed full catch-up growth within the first month of postnatal life. DEX-treated male offspring in adulthood selectively displayed elevated plasma adrenocorticotropic hormone (by 221%) and corticosterone (by 188%, P < 0.05), postprandial insulin-glucose ratios (by 100%, P < 0.05), and hepatic expression of the gluconeogenic enzyme phospho enolpyruvate carboxykinase (by 38%, P < 0.05). Conversely, DEX-programmed females were hypertensive (by 11%, P < 0.05), with elevated hepatic angiotensinogen mRNA expression (by 9%, P < 0.05), plasma angiotensinogen (by 61%, P < 0.05), and renin activity (by 88%, P < 0.05). These findings demonstrate that prenatal glucocorticoids program adulthood cardiovascular and metabolic physiology in a gender-specific pattern, and that an activated RAS may in part underlie the hypertension associated with prenatal DEX programming.

Metabolism of glucocorticoids to A-ring-reduced dihydro- and tetrahydro-derivatives by means of hepatic 5alpha- and 5beta-reductases has long been regarded as a pathway of irreversible inactivation. However, 5alpha-reduced metabolites of other steroids, e.g. testosterone and aldosterone, have significant biological activity. We investigated whether 5alpha-reduced metabolites of corticosterone are glucocorticoid receptor (GR) agonists. Corticosterone, 5alpha-tetrahydrocorticosterone (5alphaTHB), and 5alpha-dihydrocorticosterone (5alphaDHB) were similarly effective in displacing tritiated dexamethasone from binding sites in hepatocytes, whereas 5beta-reduced metabolites were less effective in binding. 5alphaTHB had glucocorticoid receptor agonist effects in vitro and in vivo. After transient co-transfection of hGR and a murine mammary tumor virus-luciferase reporter into HeLa cells, 5alphaTHB was active to a comparable extent as corticosterone (28-fold versus 37-fold induction, respectively, at 1 microm) and additive to the effect of corticosterone. 5beta-Reduced metabolites did not activate GR. In H4IIE hepatoma cells, both 5alphaTHB and corticosterone induced mRNA expression of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (6.0-versus 10.1-fold and 3.5-versus 3.9-fold at 1 microM, respectively), an effect that was inhibited by RU486. To assess in vivo glucocorticoid activity, suppression of plasma ACTH was demonstrated in adrenalectomized rats after intraperitoneal administration of vehicle (ACTH trough 80.2 pm), corticosterone (5 mg/kg; 22 pm, p < 0.001) or 5alphaTHB (5 mg/kg; 51.3 pm, p < 0.005). Similar endogenous concentrations of corticosterone and 5alphaTHB were detected in rat liver homogenates by gas chromatography mass spectrometry. We conclude that 5alpha-reduced glucocorticoids bind to and activate GR. Transcription of glucocorticoid-regulated genes in tissues that express 5alpha-reductases will thus be influenced by intracellular levels of both corticosterone and its 5alpha-reduced metabolites.

Obesity is closely associated with the metabolic syndrome, a combination of disorders including insulin resistance, diabetes, dyslipidemia, and hypertension. A role for local glucocorticoid reamplification in obesity and the metabolic syndrome has been suggested. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) regenerates active cortisol from inactive 11-keto forms, and aP2-HSD1 mice with relative transgenic overexpression of this enzyme in fat cells develop visceral obesity with insulin resistance and dyslipidemia. Here we report that aP2-HSD1 mice also have high arterial blood pressure (BP). The mice have increased sensitivity to dietary salt and increased plasma levels of angiotensinogen, angiotensin II, and aldosterone. This hypertension is abolished by selective angiotensin II receptor AT-1 antagonist at a low dose that does not affect BP in non-Tg littermates. These findings suggest that activation of the circulating renin-angiotensin system (RAS) develops in aP2-HSD1 mice. The long-term hypertension is further reflected by an appreciable hypertrophy and hyperplasia of the distal tubule epithelium of the nephron, resembling salt-sensitive or angiotensin II-mediated hypertension. Taken together, our findings suggest that overexpression of 11beta-HSD1 in fat is sufficient to cause salt-sensitive hypertension mediated by an activated RAS. The potential role of adipose 11beta-HSD1 in mediating critical features of the metabolic syndrome extends beyond obesity and metabolic complications to include the most central cardiovascular feature of this disorder.

We set out to determine whether glucocorticoid receptor activity is affected mainly by genetic or environmental factors. The affinity and capacity of the glucocorticoid receptor was measured using dexamethasone binding in whole leukocytes from 53 monozygotic and 48 dizygotic twin pairs. Receptor binding characteristics assayed from twin pairs on the same day were highly correlated within twin pairs irrespective of zygosity. Apparent Kd was negatively correlated with environmental temperature (R(2)=0.13, P0.0001) but this did not confound the intra-pair correlation, suggesting a strong familial component independent of zygosity. Receptor binding parameters were not more closely correlated in monozygotic twins than dizygotic twin pairs indicating that there is no major genetic contribution to receptor binding and that environmental influences predominate. The close similarity in binding between twin pairs in adulthood raises the possibility that familial, non-genetic, factors such as shared early life environment may programme the glucocorticoid receptor.

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase ( CYP11B2) and 11β-hydroxylase ( CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2..The phenotype of MHS CYP11B2alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11β-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [14C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher ( P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS × MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.

A polyglutamine repeat in the N-terminus of the rat glucocorticoid receptor shows polymorphism, with variants of Q2RQ5, Q2RQ15-21. We investigated whether these natural polymorphisms affect receptor function, and whether alleles with polyglutamine repeats shorter than Q2RQ5, between Q2RQ6-14, or longer than Q2RQ21 are not found naturally because they encode a dysfunctional receptor. Ligand binding and transactivation properties of sets of natural (Q2RQ5-Q2RQ21) and artificial (Q4-Q80) alleles were compared following expression in CV-1 cells. The sequence of artificial alleles at sites flanking the repeat region was altered slightly to facilitate cloning. Western blotting showed that all constructs expressed GR protein in CV-1 cells. When co-expressed with an MMTV-lacZ reporter plasmid, all GR proteins were shown to be transcriptionally active in the presence of hormone. Scatchard analysis of ligand binding curves showed that affinities for dexamethasone and corticosterone were not affected by variation in the polyglutamine repeat either the natural or artificial sets of alleles. However, affinities were greater for the artificial compared with the natural alleles (2-3-fold for dexametasone, p0.001; and 4-fold for corticosterone,p0.001). These differences provide evidence of a direct or indirect interaction within GR between the ligand binding domain and residues flanking the polyglutamine repeat of the N-terminal domain.