5α-Reductases irreversibly catalyse A-ring reduction of pregnene steroids, including glucocorticoids and androgens. Genetic disruption of 5α-reductase 1 in male mice impairs glucocorticoid clearance and predisposes to glucose intolerance and hepatic steatosis upon metabolic challenge. However, it is unclear whether this is driven by changes in androgen and/or glucocorticoid action. Female mice with transgenic disruption of 5α-reductase 1 (5αR1-KO) were studied, representing a ‘low androgen’ state. Glucocorticoid clearance and stress responses were studied in mice aged 6 months. Metabolism was assessed in mice on normal chow (aged 6 and 12 m) and also in a separate cohort following 1-month high-fat diet (aged 3 m). Female 5αR1-KO mice had adrenal suppression (44% lower AUC corticosterone after stress), and upon corticosterone infusion, accumulated hepatic glucocorticoids (~27% increased corticosterone). Female 5αR1-KO mice aged 6 m fed normal chow demonstrated insulin resistance (~35% increased area under curve (AUC) for insulin upon glucose tolerance testing) and hepatic steatosis (~33% increased hepatic triglycerides) compared with controls. This progressed to obesity (~12% increased body weight) and sustained insulin resistance (~38% increased AUC insulin) by age 12 m. Hepatic transcript profiles supported impaired lipid β-oxidation and increased triglyceride storage. Female 5αR1-KO mice were also predisposed to develop high-fat diet-induced insulin resistance. Exaggerated predisposition to metabolic disorders in female mice, compared with that seen in male mice, after disruption of 5αR1 suggests phenotypic changes may be underpinned by altered metabolism of glucocorticoids rather than androgens.
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Dawn E W Livingstone, Emma M Di Rollo, Tracy C-S Mak, Karen Sooy, Brian R Walker and Ruth Andrew
Dawn E W Livingstone, Emma M Di Rollo, Chenjing Yang, Lucy E Codrington, John A Mathews, Madina Kara, Katherine A Hughes, Christopher J Kenyon, Brian R Walker and Ruth Andrew
Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease.
Lesley A Hill, Dimitra A Vassiliadi, Ioanna Dimopoulou, Anna J Anderson, Luke D Boyle, Alixe H M Kilgour, Roland H Stimson, Yoan Machado, Christopher M Overall, Brian R Walker, John G Lewis and Geoffrey L Hammond
Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.
Sara Della Torre, Gianpaolo Rando, Clara Meda, Paolo Ciana, Luisa Ottobrini and Adriana Maggi
Oestrogens are well-known proliferation and differentiation factors that play an essential role in the correct development of sex-related organs and behaviour in mammals. With the use of the ERE-Luc reporter mouse model, we show herein that throughout mouse development, oestrogen receptors (ERs) are active starting from day 12 post conception. Most interestingly, we show that prenatal luciferase expression in each organ is proportionally different in relation to the germ layer of the origin. The luciferase content is highest in ectoderm-derived organs (such as brain and skin) and is lowest in endoderm-derived organs (such as liver, lung, thymus and intestine). Consistent with the testosterone surge occurring in male mice at the end of pregnancy, in the first 2 days after birth, we observed a significant increase in the luciferase content in several organs, including the liver, bone, gonads and hindbrain. The results of the present study show a widespread transcriptional activity of ERs in developing embryos, pointing to the potential contribution of these receptors in the development of non-reproductive as well as reproductive organs. Consequently, the findings reported here might be relevant in explaining the significant differences in male and female physiopathology reported by a growing number of studies and may underline the necessity for more systematic analyses aimed at the identification of the prenatal effects of drugs interfering with ER signalling, such as aromatase inhibitors or endocrine disrupter chemicals.
E J Agnew, A Garcia-Burgos, R V Richardson, H Manos, A J W Thomson, K Sooy, G Just, N Z M Homer, C M Moran, P J Brunton, G A Gray and K E Chapman
Endogenous glucocorticoid action is important in the structural and functional maturation of the fetal heart. In fetal mice, although glucocorticoid concentrations are extremely low before E14.5, glucocorticoid receptor (GR) is expressed in the heart from E10.5. To investigate whether activation of cardiac GR prior to E14.5 induces precocious fetal heart maturation, we administered dexamethasone in the drinking water of pregnant dams from E12.5 to E15.5. To test the direct effects of glucocorticoids upon the cardiovascular system we used SMGRKO mice, with Sm22-Cre-mediated disruption of GR in cardiomyocytes and vascular smooth muscle. Contrary to expectations, echocardiography showed no advancement of functional maturation of the fetal heart. Moreover, litter size was decreased 2 days following cessation of antenatal glucocorticoid exposure, irrespective of fetal genotype. The myocardial performance index and E/A wave ratio, markers of fetal heart maturation, were not significantly affected by dexamethasone treatment in either genotype. Dexamethasone treatment transiently decreased the myocardial deceleration index (MDI; a marker of diastolic function), in control fetuses at E15.5, with recovery by E17.5, 2 days after cessation of treatment. MDI was lower in SMGRKO than in control fetuses and was unaffected by dexamethasone. The transient decrease in MDI was associated with repression of cardiac GR in control fetuses following dexamethasone treatment. Measurement of glucocorticoid levels in fetal tissue and hypothalamic corticotropin-releasing hormone (Crh) mRNA levels suggest complex and differential effects of dexamethasone treatment upon the hypothalamic–pituitary–adrenal axis between genotypes. These data suggest potentially detrimental and direct effects of antenatal glucocorticoid treatment upon fetal heart function.
Katie J Mylonas, Neil A Turner, Sumia A Bageghni, Christopher J Kenyon, Christopher I White, Kieran McGregor, Robert A Kimmitt, Richard Sulston, Valerie Kelly, Brian R Walker, Karen E Porter, Karen E Chapman and Gillian A Gray
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.
Tingting Yang, Min He, Hailiang Zhang, Paula Barrett and Changlong Hu
Aldosterone, which plays a key role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal cortex. Exaggerated overproduction of aldosterone from ZG cells causes primary hyperaldosteronism. In ZG cells, calcium entry through voltage-gated calcium channels plays a central role in the regulation of aldosterone secretion. Previous studies in animal adrenals and human adrenocortical cell lines suggest that the T-type but not the L-type calcium channel activity drives aldosterone production. However, recent clinical studies show that somatic mutations in L-type calcium channels are the second most prevalent cause of aldosterone-producing adenoma. Our objective was to define the roles of T and L-type calcium channels in regulating aldosterone secretion from human adrenals. We find that human adrenal ZG cells mainly express T-type CaV3.2 /3.3 and L-type CaV1.2/1.3 calcium channels. TTA-P2, a specific inhibitor of T-type calcium channel subtypes, reduced basal aldosterone secretion from acutely prepared slices of human adrenals. Surprisingly, nifedipine, the prototypic inhibitor of L-type calcium channels, also decreased basal aldosterone secretion, suggesting that L-type calcium channels are active under basal conditions. In addition, TTA-P2 or nifedipine also inhibited aldosterone secretion stimulated by angiotensin II- or elevations in extracellular K+. Remarkably, blockade of either L- or T-type calcium channels inhibits basal and stimulated aldosterone production to a similar extent. Low concentrations of TTA-P2 and nifedipine showed additive inhibitory effects on aldosterone secretion. We conclude that T- and L-type calcium channels play equally important roles in controlling aldosterone production from human adrenals.