49 research outputs found
Dysregulation of glucocorticoid metabolism in murine obesity: comparable effects of leptin resistance and deficiency
In obese humans, metabolism of glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and A-ring reduction (by 5α- and 5β-reductases) is dysregulated in a tissue specific manner. These changes have been recapitulated in leptin resistant obese Zucker rats but were not observed in high-fat fed Wistar rats. Recent data from mouse models suggest that such discrepancies may reflect differences in leptin signalling. We therefore compared glucocorticoid metabolism in murine models of leptin deficiency and resistance. Male ob/ob and db/db mice and their respective littermate controls (n=10–12/group) were studied at the age of 12 weeks. Enzyme activities and mRNA expression were quantified in snap-frozen tissues. The patterns of altered pathways of steroid metabolism in obesity were similar in ob/ob and db/db mice. In liver, 5β-reductase activity and mRNA were increased and 11β-HSD1 decreased in obese mice, whereas 5α-reductase 1 (5αR1) mRNA was not altered. In visceral adipose depots, 5β-reductase was not expressed, 11β-HSD1 activity was increased and 5αR1 mRNA was not altered in obesity. By contrast, in subcutaneous adipose tissue 11β-HSD1 and 5αR1 mRNA were decreased. Systematic differences were not found between ob/ob and db/db murine models of obesity, suggesting that variations in leptin signalling through the short splice variant of the Ob receptor do not contribute to dysregulation of glucocorticoid metabolism
5α-TETRAHYDROCORTICOSTERONE: A TOPICAL ANTI-INFLAMMATORY GLUCOCORTICOID WITH AN IMPROVED THERAPEUTIC INDEX IN A MURINE MODEL OF DERMATITIS
Background and PurposeGlucocorticoids are powerful anti-inflammatory drugs, but are associated with many side-effects. Topical application in atopic dermatitis leads to skin thinning, metabolic changes, and adrenal suppression. 5α-Tetrahydrocorticosterone (5αTHB) is a potential selective anti-inflammatory with reduced metabolic effects. Here, the efficacy and side-effect profile of 5αTHB were compared with hydrocortisone in preclinical models of irritant dermatitis.Experimental ApproachAcute irritant dermatitis was invoked in ear skin of male C57BL/6 mice with a single topical application of croton oil. Inflammation was assessed as oedema via ear weight following treatment with 5αTHB and hydrocortisone. Side-effects of 5αTHB and hydrocortisone were assessed following chronic topical steroid treatment (28 days) to non-irritated skin. Skin thinning was quantified longitudinally by caliper measurements and summarily by qPCR for transcripts for genes involved in extracellular matrix homeostasis; systemic effects of topical steroid administration also were assessed. Clearance of 5αTHB and hydrocortisone were measured following intravenous and oral administration.Key Results5αTHB suppressed ear swelling in mice, with ED50 similar to hydrocortisone (23 μg vs. 13 μg). Chronic application of 5αTHB did not cause skin thinning, adrenal atrophy, weight loss, thymic involution, or raised insulin levels, all of which were observed with topical hydrocortisone. Transcripts for genes involved in collagen synthesis and stability were adversely affected by all doses of hydrocortisone, but only by the highest dose of 5αTHB (8× ED50). 5αTHB was rapidly cleared from the systemic circulation.Conclusions and ImplicationsTopical 5αTHB has potential to treat inflammatory skin conditions, particularly in areas of delicate skin
Mid-IR spectroscopic instrumentation for point-of-care diagnosis using a hollow silica waveguide gas cell
Laser spectroscopy provides the basis of instrumentation developed for the diagnosis of infectious disease, via
quantification of organic biomarkers that are produced by associated bacteria. The technology is centred on a multichannel
pulsed quantum cascade laser system that allows multiple lasers with different wavelengths to be used
simultaneously, each selected to monitor a different diagnostic biomarker. The instrument also utilizes a hollow silica
waveguide (HSW) gas cell which has a very high ratio of interaction pathlength to internal volume. This allows sensitive
detection of low volume gas species from small volume biological samples. The spectroscopic performance of a range of
HSW gas cells with different lengths and bore diameters has been assessed using methane as a test gas and a best-case
limit of detection of 0.26 ppm was determined. The response time of this cell was measured as a 1,000 sccm flow of
methane passed through it and was found to be 0.75 s. These results are compared with those obtained using a multi-pass
Herriot cell. A prototype instrument has been built and approved for clinical trials for detection of lung infection in
acute-care patients via analysis of ventilator breath. Demonstration of the instrument for headspace gas analysis is made
by monitoring the methane emission from bovine faeces. The manufacture of a hospital-ready device for monitoring
biomarkers of infection in the exhaled breath of intensive care ventilator patients is also presented
Metformin increases cortisol regeneration by 11βHSD1 in obese men with and without type 2 diabetes mellitus
CONTEXT:The mechanism of action of metformin remains unclear. Given the regulation of the cortisol-regenerating enzyme 11βhydroxysteroid dehydrogenase 1 (11βHSD1) by insulin and the limited efficacy of selective 11βHSD1 inhibitors to lower blood glucose when co-prescribed with metformin, we hypothesized that metformin reduces 11βHSD1 activity.OBJECTIVE:To determine whether metformin regulates 11βHSD1 activity in vivo in obese men with and without type 2 diabetes mellitus.DESIGN:Double-blind, randomized, placebo-controlled, crossover study.SETTING:A hospital clinical research facility.PARTICIPANTS:Eight obese nondiabetic (OND) men and eight obese men with type 2 diabetes (ODM).INTERVENTION:Participants received 28 days of metformin (1 g twice daily), placebo, or (in the ODM group) gliclazide (80 mg twice daily) in random order. A deuterated cortisol infusion at the end of each phase measured cortisol regeneration by 11βHSD1. Oral cortisone was given to measure hepatic 11βHSD1 activity in the ODM group. The effect of metformin on 11βHSD1 was also assessed in human hepatocytes and Simpson-Golabi-Behmel syndrome adipocytes.MAIN OUTCOME MEASURES:The effect of metformin on whole-body and hepatic 11βHSD1 activity.RESULTS:Whole-body 11βHSD1 activity was approximately 25% higher in the ODM group than the OND group. Metformin increased whole-body cortisol regeneration by 11βHSD1 in both groups compared with placebo and gliclazide and tended to increase hepatic 11βHSD1 activity. In vitro, metformin did not increase 11βHSD1 activity in hepatocytes or adipocytes.CONCLUSIONS:Metformin increases whole-body cortisol generation by 11βHSD1 probably through an indirect mechanism, potentially offsetting other metabolic benefits of metformin. Co-prescription with metformin should provide a greater target for selective 11βHSD1 inhibitors
Aromatase Inhibition Reduces Insulin Sensitivity in Healthy Men
CONTEXT: Deficiency of aromatase, the enzyme that catalyzes the conversion of androgens to estrogens, is associated with insulin resistance in humans and mice. OBJECTIVE: We hypothesized that pharmacological aromatase inhibition results in peripheral insulin resistance in humans. DESIGN: This was a double-blind, randomized, controlled, crossover study. SETTING: The study was conducted at a clinical research facility. PARTICIPANTS: Seventeen healthy male volunteers (18–50 y) participated in the study. INTERVENTION: The intervention included oral anastrozole (1 mg daily) and placebo, each for 6 weeks with a 2-week washout period. MAIN OUTCOME MEASURE: Glucose disposal and rates of lipolysis were measured during a stepwise hyperinsulinemic euglycemic clamp. Data are mean (SEM). RESULTS: Anastrozole therapy resulted in significant estradiol suppression (59.9 ± 3.6 vs 102.0 ± 5.7 pmol/L, P = < .001) and a more modest elevation of total T (25.8 ± 1.2 vs 21.4 ± 0.7 nmol/L, P = .003). Glucose infusion rate, during the low-dose insulin infusion, was lower after anastrozole administration (12.16 ± 1.33 vs 14.15 ± 1.55 μmol/kg·min, P = .024). No differences in hepatic glucose production or rate of lipolysis were observed. CONCLUSION: Aromatase inhibition reduces insulin sensitivity, with respect to peripheral glucose disposal, in healthy men. Local generation and action of estradiol, at the level of skeletal muscle, is likely to be an important determinant of insulin sensitivity
11β-HSD2 SUMOylation Modulates Cortisol-induced Mineralocorticoid Receptor Nuclear Translocation Independently of Effects on Transactivation
The enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) has an essential role in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor (MR) by converting 11β-hydroxyglucocorticoids to inactive 11-ketosteroids. Congenital deficiency of 11β-HSD2 causes a form of salt-sensitive hypertension known as the syndrome of apparent mineralocorticoid excess. The disease phenotype, which ranges from mild to severe, correlates well with reduction in enzyme activity. Furthermore, polymorphisms in the 11β-HSD2 coding gene (HSD11B2) have been linked to high blood pressure and salt sensitivity, major cardiovascular risk factors. 11β-HSD2 expression is controlled by different factors such as cytokines, sex steroids, or vasopressin, but posttranslational modulation of its activity has not been explored. Analysis of 11β-HSD2 sequence revealed a consensus site for conjugation of small ubiquitin-related modifier (SUMO) peptide, a major posttranslational regulatory event in several cellular processes. Our results demonstrate that 11β-HSD2 is SUMOylated at lysine 266. Non-SUMOylatable mutant K266R showed slightly higher substrate affinity and decreased Vmax, but no effects on protein stability or subcellular localization. Despite mild changes in enzyme activity, mutant K266R was unable to prevent cortisol-dependent MR nuclear translocation. The same effect was achieved by coexpression of wild-type 11β-HSD2 with sentrin-specific protease 1, a protease that catalyzes SUMO deconjugation. In the presence of 11β-HSD2-K266R, increased nuclear MR localization did not correlate with increased response to cortisol or increased recruitment of transcriptional coregulators. Taken together, our data suggests that SUMOylation of 11β-HSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation
Spatial Localization and Quantitation of Androgens in Mouse Testis by Mass Spectrometry Imaging
Androgens
are essential for male development and reproductive function.
They are transported to their site of action as blood-borne endocrine
hormones but can also be produced within tissues to act in intracrine
and paracrine fashions. Because of this, circulating concentrations
may not accurately reflect the androgenic influence within specific
tissue microenvironments. Mass spectrometry imaging permits regional
analysis of small molecular species directly from tissue surfaces.
However, due to poor ionization and localized ion suppression, steroid
hormones are difficult to detect. Here, derivatization with Girard
T reagent was used to charge-tag testosterone and 5α-dihydrotestosterone
allowing direct detection of these steroids in mouse testes, in both
basal and maximally stimulated states, and in rat prostate. Limits
of detection were ∼0.1 pg for testosterone. Exemplary detection
of endogenous steroids was achieved by matrix-assisted laser desorption
ionization and either Fourier transform ion cyclotron resonance detection
(at 150 μm spatial resolution) or quadrupole-time-of-flight
detection (at 50 μm spatial resolution). Structural confirmation
was achieved by collision induced fragmentation following liquid extraction
surface analysis and electrospray ionization. This application broadens
the scope for derivatization strategies on tissue surfaces to elucidate
local endocrine signaling in health and disease