Mineralocorticoid Receptor mRNA Expression Research Articles

Abstract 1110: The Sex-specific Role Of The Mineralocorticoid Receptor In Smooth Muscle Cells In Obesity-associated Vascular Stiffness

Introduction: Obesity is associated with increased vascular stiffness which predicts risk for heart attack, stroke and cardiovascular (CV) mortality. Obesity is more common and has a greater negative impact on CV health in women. Perivascular adipose tissue (PVAT), which contributes to maintenance of normal vascular tone, becomes dysfunctional in obesity, but its role in vascular stiffness is less well studied. Mineralocorticoid receptor (MR) is a hormone-activated transcription factor regulated by inflammation and oxidative stress in aging vascular smooth muscle cells (SMC) where it induces vascular stiffness. The role of SMC-MR in obesity-associated vascular stiffness and whether there are sex differences in the mechanism is unknown. Hypothesis: We hypothesized that sex differences in obesity-induced changes in PVAT alters aortic inflammation and oxidative stress (OS) to induce SMC MR and contribute to aortic stiffness by regulating sexually dimorphic gene programs. Methods and Results: Young male and female mice (3 months old) with MR intact or SMC-MR knocked out (SMC-MR-KO) were fed with high-fat diet (HFD) for 3 months. In both sexes, MR mRNA expression increased with obesity in MR intact mice, as did aortic stiffness (by pulse wave velocity (PWV)). SMC-MR-KO prevented the HFD-induced vascular stiffness in both sexes. In aortas from male mice only, NFkB protein were induced by obesity, independent of the MR. Obese males had increased vascular fibrosis histologically, vascular fibrosis gene (Col1, CTGF) expression and inflammation (TNFa, IL1B) in MR intact mice and is prevented in the SMC-MR-KO mice. In females, HIF1a protein and mRNA were induced by obesity, independent of SMC MR. In MR-intact females, OS gene expression (NADPH-oxidase 2-4 (Nox2, Nox4)) was increased by HFD. Human aortic SMC (HASMCs) from lean/obese male and female donors were cultured. NFkB, MR, and Col1 were increased with obesity in HASMCs from males while HIF1a, MR, Nox2 and Nox4 were increased in obese female donors compared with non-obese. Conclusions: These data support sexually dimorphic, SMC MR-dependent mechanisms for vascular stiffness associated with obesity and suggest that sex-specific therapies may be needed to prevent obesity-associated vascular stiffness.

Down-Regulation of the Mineralocorticoid Receptor (MR) and Up-Regulation of Hydroxysteroid 11-Beta Dehydrogenase Type 2 (HSD11B2) Isoenzyme in Critically Ill Patients.

Objective: The mineralocorticoid receptor (MR) has two ligands, aldosterone and cortisol. Hydroxysteroid 11-beta dehydrogenase (HSD11B) isoenzymes regulate which ligand will bind to MR. In this study we aimed to evaluate the expression of the MR and the HSD11B isozymes in peripheral polymorphonuclear cells (PMNs) in critical illness for a 13-day period.Design: Prospective studySetting: One multi-disciplinary intensive care unit (ICU)Participants: Forty-two critically ill patientsMethods: Messenger RNA (mRNA) expression of MR, HSD11B1, and HSD11B2, aldosterone levels, and plasma renin activity (PRA) were measured in 42 patients on ICU admission and on days 4, 8, and 13. Twenty-five age and sex-matched healthy subjects were used as controls.Results: Compared to healthy controls, MR expression in critically ill patients was lower during the entire study period. HSD11B1 expression was also lower, while HSD11B2 expression was higher. In patients, PRA, aldosterone, the aldosterone:renin ratio, and cortisol remained unaltered during the study period.Conclusion: Our results suggest that, in our cohort of critically ill patients, local endogenous cortisol availability is diminished, pointing towards glucocorticoid resistance. Aldosterone probably occupies the MR, raising the possibility that PMNs might be useful to study to gain insights into MR functionality during pathological states.

Effect of maternal periconceptional undernutrition in sheep on cortisol regulation in offspring from mid-late gestation, through to adulthood.

Maternal periconceptional undernutrition (PCUN) alters fetal hypothalamic-pituitary-adrenal axis (HPAA) function and placental glucocorticoid metabolism in sheep. The effects of PCUN on HPAA function in adult life are not known. We investigated the effects of PCUN on fetal adrenal development across gestation and on cortisol regulation in adult offspring. Ewes were undernourished from 61 days before to 30 days after conception ('PCUN') or fed ad libitum ('N'). mRNA expression in the fetal adrenal gland of ACTH receptor (ACTHR), steroidogenic acute regulatory protein (STAR), cytochrome P450 17A1 (CYP17A1), 11beta-hydroxysteroid-dehydrogenase type 2 (11βHSD2), insulin-like growth factor-2 (IGF2), and in the fetal hippocampus of 11βHSD1, 11βHSD2, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) was determined at 50 (adrenal only), 85, 120 and 131 days of gestation (term=148 days). In adult offspring (≥ 3 years, N; 10 female, 5 male, PCUN; 10 female, 10 male) a combined arginine vasopressin (AVP, 0.1 μg/kg) and corticotropin-releasing hormone (CRH, 0.5 μg/kg) challenge and a metyrapone (40 mg/kg) challenge were undertaken. mRNA expression of ACTHR, STAR and CYP17A1 were determined in adult adrenals. Fetal adrenal STAR, CYP17A1 and IGF2 mRNA expression were not different between groups in early gestation but were higher in PCUN than N at 131 days' gestation (all p<0.01). PCUN reduced fetal hippocampal MR and GR mRNA expression by 50% at 85 day, but not in later gestation. Adult offspring plasma cortisol responses to AVP+CRH or metyrapone were not different between groups. Plasma ACTH response to AVP+CRH was lower in PCUN males but ACTH response to metyrapone was not different between groups. Adult adrenal ACTHR, STAR, and CYP17A1 mRNA expression were not affected by PCUN. We conclude that the effects of PCUN on fetal HPAA function that became apparent in late gestation, are not reflected in adrenal cortisol secretion in mid-adulthood.

Juvenile social isolation affects the structure of the tanycyte–vascular interface in the hypophyseal portal system of the adult mice

Accumulating evidence indicates that social stress in the juvenile period affects hypothalamic–pituitary–adrenal (HPA) axis activity in adulthood. The biological mechanisms underlying this phenomenon remain unclear. We aimed to elucidate them by comparing adult mice that had experienced social isolation from postnatal day 21–35 (juvenile social isolation (JSI) group) with those reared normally (control group). JSI group mice showed an attenuated HPA response to acute swim stress, while the control group had a normal response to this stress. Activity levels of the paraventricular nucleus in both groups were comparable, as shown by c-Fos immunoreactivities and mRNA expression of c-Fos, Corticotropin-releasing factor (CRF), Glucocorticoid receptor, and Mineralocorticoid receptor. We found greater vascular coverage by tanycytic endfeet in the median eminence of the JSI group mice than in that of the control group mice under basal condition and after acute swim stress. Moreover, CRF content after acute swim stress was greater in the median eminence of the JSI group mice than in that of the control group mice. The attenuated HPA response to acute swim stress was specific to JSI group mice, but not to control group mice. Although a direct link awaits further experiments, tanycyte morphological changes in the median eminence could be related to the HPA response.

Lactation overnutrition-induced obesity impairs effects of exogenous corticosterone on energy homeostasis and hypothalamic-pituitary-adrenal axis in male rats

AimsLitter size reduction on the first days of life results in increased body weight and adiposity, with higher levels of circulating glucocorticoids. Obese rodents are more sensitive to the anabolic effects of glucocorticoids and less responsive to glucocorticoids feedback on hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to evaluate effects of the treatment with corticosterone on metabolic responses and HPA axis in adult male rats reared in small litters. Main methodsFrom postnatal day (PND) 60 to 88, adult male rats of normal (NL- 10 pups/dam) and small (SL- 3 pups/dam) litters received oral treatment with Corticosterone (CORT-15 mg/L) in the drinking water or no treatment, composing the four experimental groups (NL-water; NL-CORT; SL-water and SL-CORT), for the evaluation of energy homeostasis and HPA axis. Key findingsMale rats of SL-water group presented on PND88: glucose intolerance, higher adiposity, plasma triglycerides, free fatty acids, total and low-density lipoprotein (LDL) cholesterol and corticosterone. SL-water animals showed increased mRNA of corticotrophin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) and proopiomelanocortin (POMC) in the pituitary, with decreased mRNA expression of PVN mineralocorticoid receptor. NL-CORT animals presented glucose intolerance, increased body weight, food intake, total and LDL cholesterol. Glucocorticoid treatment reduced corticosterone levels and adrenal cortex thickness in NL group, associated with increased mRNA of PVN CRH and pituitary POMC, without effects on SL animals. SignificanceLactation overnutrition promotes hyperreactivity of HPA axis and reduces the responsiveness to glucocorticoids effects on energy balance and negative feedback of HPA axis in adult male rats.

Effect of wheat-grain moxibustion on the expression of 5-HT and cortisol in the serum, and MR and GR in the hippocampus in rats with hypothyroidism complicated with depression

To observe the effect of wheat-grain moxibustion on behavior, 5-hydroxytryptamine (5-HT) and cortisol in the serum, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hippocampus in rats with hypothyroidism complicated with depression, and to explore the possible mechanism of wheat-grain moxibustion on improving depression in rats with hypothyroidism. A total of 32 SPF SD rats were randomly divided into a blank group, a model group, a medication group and a wheat-grain moxibustion group, 8 rats in each group. Except for the blank group, the rats in the remaining groups were treated with intragastric administration of 0.1% propylthiouracil (PTU) suspension at 1 mL/100 g, once a day for 4 weeks to establish the rat model of hypothyroidism, and whether the rats were accompanied with depression-like behavior determined through behavioristics evaluation. The rats in the medication group were intervened with euthyrox at 0.9 mL/100 g, once a day, for 4 weeks; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Mingmen" (GV 4), "Shenshu" (BL 23) and "Pishu" (BL 20), 7 cones each acupoint, once a day, six times a week for 4 weeks. After the intervention, the depression status was observed by behavioristics test; the contents of thyroid stimulating hormone (TSH), total thyroxine (TT4), 5-HT and cortisol in the serum were detected by ELISA; the protein expressions of MR and GR in hippocampus were detected by Western blot; the expressions of MR mRNA and GR mRNA in the hippocampus were detected by real-time PCR. Before the intervention, compared with the blank group, the scores of open field test (OFT) were decreased and the immobility time of tail suspension test (TST) was prolonged (P<0.05); the serum TSH contents were increased and TT4 contents were decreased (P<0.01) in the other three groups. After the intervention, compared with the model group, the vertical score of OFT was increased and the immobility time of forced swimming test (FST) was prolonged in the medication group (P<0.05), while the scores of three items of OFT were increased (P<0.05, P<0.01), and the immobility time of FST and TST was shortened in the wheat-grain moxibustion group (P<0.01, P<0.05). Compared with the medication group, the immobility time of TST and FST in the wheat-grain moxibustion group was shorter (P<0.05, P<0.01). Compared with the blank group, in the model group, the contents of serum TSH and cortisol were increased (P<0.01, P<0.001), while the contents of serum TT4 and 5-HT were decreased (P<0.01, P<0.001). Compared with the model group, the contents of serum TT4 and 5-HT were increased, while the contents of serum TSH and cortisol were decreased in the medication group and wheat-grain moxibustion group (P<0.01, P<0.05). Compared with the blank group, the protein and mRNA expression of MR, GR in the hippocampus in the model group was decreased (P<0.01, P<0.05, P<0.001); compared with the model group, the protein and mRNA expression of MR in the hippocampus in the medication group were increased (P<0.05), and the protein expression of MR, GR and mRNA expression of MR in the hippocampus in the wheat-grain moxibustion group were increased (P<0.05, P<0.01). Compared with the medication group, the expression of MR mRNA in the wheat-grain moxibustion group was increased (P<0.05). Wheat-grain moxibustion could significantly improve thyroid function and depression in rats with hypothyroidism. Its mechanism may be related to up-regulating the protein and mRNA expression of MR and GR in the hippocampus, and then affecting the expression of serum cortisol and 5-HT.

Hypothalamic-pituitary-adrenal axis regulation and organization in urban and rural song sparrows

Urban habitats present animals with persistent disturbances and acute stressors not present in rural habitats or present at significantly lower levels. Differences in the glucocorticoid stress response could underlie colonization of these novel habitats. Despite urban habitats characterization as more stressful, previous comparisons of urban and rural birds have failed to find consistent differences in baseline and stress induced glucocorticoid levels. Another aspect of glucocorticoid regulation that could underlie an animal’s ability to inhabit novel habitats, but has yet to be well examined, is more efficient termination of the glucocorticoid stress response which would allow birds in urban habitats to recover more quickly after a disturbance. The glucocorticoid stress response is terminated by negative feedback achieved primarily through their binding of receptors in the hippocampus and hypothalamus and subsequent decreased synthesis and release from the adrenals. We investigated if male song sparrows (Melospiza melodia) in urban habitats show more efficient termination of the glucocorticoid stress response than their rural counterparts using two approaches. First, we measured glucocorticoid receptor, mineralocorticoid receptor and 11β-HSD2 (an enzyme that inactivates corticosterone) mRNA expression in negative feedback targets of the brain (the hippocampus and hypothalamus) as a proxy measure of sensitivity to negative feedback. Second, we measured plasma corticosterone levels after standardized restraint and again following a challenge with the synthetic glucocorticoid, dexamethasone, as a means of assessing how quickly birds decreased glucocorticoid synthesis and release. Though there were no differences in the hypothalamus of urban and rural song sparrows, urban birds had lower glucocorticoid receptor and 11β-HSD2 mRNA expression in the hippocampus. Further, urban and rural birds had similar reductions in corticosterone following the dexamethasone challenge, suggesting that they do not differ in how quickly they decrease glucocorticoid synthesis and release. Thus, urban and rural song sparrows display similar termination of the glucocorticoid stress response even though urban birds have decreased hippocampal glucocorticoid receptor and 11β-HSD2 abundance.

Anabolic steroid excess promotes hydroelectrolytic and autonomic imbalance in adult male rats: Is it enough to alter blood pressure?

AimThe present study investigated the effects of anabolic steroid (AS) excess on blood pressure regulation. MethodsMale Wistar rats were treated with nandrolone decanoate (AS) or vehicle (CTL) for 8 or 10 weeks. Saline (1.8%) and water intake were measured in metabolic cages. Urinary volume, osmolarity, Na+ and K+ concentrations, and plasma osmolarity were measured. The autonomic balance was estimated by heart rate variability at baseline or after icv injection of losartan. Cardiac function was assessed by echocardiography and ex vivo recordings. Myocardial collagen deposition was evaluated by Picrosirius-Red staining. Vascular reactivity and wall thickness were investigated in aortic sections. Blood pressure (BP) was assessed by tail-cuff plethysmography. Angiotensin II type I receptor (AT1R), renin, and mineralocorticoid receptor (MR) mRNA expression was measured in the kidneys and whole hypothalamus. ResultsAS group exhibited decreased urinary volume and Na+ concentration, while urinary K+ concentration, plasma osmolarity, and renal AT1R and renin mRNA levels were increased compared to CTL (p < 0.05). Water intake was increased, and saline intake was decreased in the AS group (p < 0.01). AS group exhibited increased low-frequency/high-frequency-ratio, while it was decreased by icv injection of losartan (p < 0.05) compared to baseline. Neither cardiac function nor vascular reactivity/morphology was affected by AS excess (p > 0.05). Ultimately, BP levels were not altered by AS excess (p > 0.05). ConclusionAS excess promoted hydroelectrolytic and autonomic imbalance but did not alter vascular or cardiac function/morphology.

Coping with extremes: Remarkably blunt adrenocortical responses to acute stress in two sympatric snow finches on the Qinghai-Tibet Plateau during winter relative to other seasons

The extreme climatic conditions (ECCs) of the Qinghai-Tibet Plateau impose strong selective pressures on the evolution of phenotypic traits in free-living animals. It is not well understood how animals on the Qinghai-Tibet Plateau modify their adrenocortical functions in response to both predictable and unpredictable events of ECCs, especially when the available resources are lowest during the wintering life-history stage. To uncover potential physiological mechanisms, we studied the life history stage dependent features of morphology, the plasma corticosterone response to acute stress and brain glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression in two sympatric snow finches: the white-rumped snow finch (Onychostruthus taczanowskii, WRSF); and the rufous-necked snow finch, Pyrgilauda ruficollis, RNSF) in Qinghai Province, China. Our results showed that (a) baseline corticosterone and stressor-induced corticosterone levels significantly varied with life history stage, but not between the species; (b) in WRSF, GR mRNA expression in the paraventricular nucleus was higher in the wintering stage compared to the pre-basic molt stage. There were no differences in hippocampus MR mRNA expression between stages in either species; (c) in the wintering stage, the suppression of corticosterone secretion in both species was an unexpected strategy in free-living animals. Both convergent and divergent phenotypic traits of adrenocortical responses to acute stress in two sympatric snow finches contribute to our understanding of the coping mechanisms of closely related species in the severe winter on the Qinghai-Tibet Plateau.

Effect of Hirsutella sinensis Fungus on the Hypothalamic-Pituitary-Adrenal Axis in Lewis Rats with Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a classic syndrome in traditional Chinese medicine, which is mainly caused by damage to the hypothalamic-pituitary-adrenal (HPA) axis. Hirsutella sinensis fungus (HSF), an artificial substitute of Cordyceps sinensis, has been widely used in TCM. However, the effects and the possible mechanism of HSF on the HPA axis and corresponding KYDS have not yet been investigated. In this study, Lewis rats were used as a spontaneous KYDS model. HSF was intragastrically administered to the Lewis rats at two doses: low dose (1 g/kg) and high dose (2 g/kg). Body weight, temperature, and behavioral tests including grip strength, open field, and Morris water maze (MWM) tests were used to evaluate the KYDS symptoms. Enzyme-linked immunosorbent assay was used to detect the level of circulating adrenocortisol (ACTH), corticosterone (CORT), corticotropin releasing hormone (CRH), cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 10 (IL-10), CRH, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) was detected by quantitative real-time polymerase chain reaction (Q-PCR). The Lewis rats were indicated to have KYDS symptoms and HSF treatment ameliorated these symptoms via enhancement of the HPA axis function, which was evidenced by the increased levels of CRH, ACTH, and CORT in serum and 17-OHCS in urine. HSF also significantly improved the expression of TNF-α, IFN-γ, and IL-2, secreted by Th1 cells, which might accelerate the activation of the immune system related to the HPA axis function. Thus, we conclude that HSF can alleviate KYDS symptoms in Lewis rats by regulating the HPA axis through accelerated immune system activation.

Prenatal Corticosterone Exposure Alters Glucocorticoid Metabolic Enzyme Eene mRNA Associated with Increased Aggressive Behaviors and Tonic Immobility in Chicken

Exposure to excess Glucocorticoids (GCs) during embryonic development influences offspring physiology and behaviors and induces change in Hypothalamic-Pituitary-Adrenal (HPA) axis genes expression and serotonergic system in mammals. Whether prenatal corticosterone (CORT) exposure induces similar effects in avian species remains unclear. In the present study, we injected low (0.2 μg) and high (1 μg) doses of CORT in ovo before incubation and detected changes in aggressive behavior, Tonic Immobility (TI), HPA axis and 5-hydroxytryptamine (serotonin) (5-HT) system gene expression on post hatch chickens of different ages. High dose of CORT significantly (P<0.05) suppressed growth rate, increased the frequency of aggressive behaviors, which was associated with elevated plasma CORT concentration. Likewise, in ovo injection of CORT significantly (P<0.05) increased Tonic Immobility (TI) duration both in chickens from low and high doses of CORT treatments compared to control. In addition, administration of CORT significantly (P<0.05) up-regulated mRNA expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) whereas it down-regulated 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and mineralocorticoid receptor (MR) mRNA expression in the hypothalamus. No significant differences were seen in Glucocorticoid Receptor (GR) and 20-hydroxysteroid dehydrogenase (20-HSD) mRNA levels upon CORT treatment. Moreover, CORT exposure significantly (P<0.05) increased hypothalamic 5-hydroxytryptamine (serotonin) receptor 1A (5-HTR1A) mRNA expression, but not 5-HT receptor 1B (5-HTR1B). In ovo administration of CORT may programs the aggressive behaviors in the chicken through alterations of HPA axis and 5-HT system.

Limited Support for Thyroid Hormone or Corticosterone Related Gene Expression as a Proximate Mechanism of Incubation Temperature-Dependent Phenotypes in Birds.

The conditions that animals experience during early development can have profound consequences for health and fitness. In birds, one of the most important aspects of development is egg incubation temperature. A small decrease in average temperature leads to various impacts on offspring phenotype, such as smaller body sizes, slower growth rates, and less efficient metabolic activity. Little is known, however, about the proximate mechanisms underlying these incubation temperature-induced phenotypic changes. Two important hormones which could play a proximate role are thyroid hormone and corticosterone, which mobilize stored energy reserves and coordinate the normal growth of tissues, particularly in the brain. Previous research shows that circulating blood concentrations of both hormones are influenced by incubation temperature, but the mechanism by which incubation temperature may lead to these changes is unknown. We hypothesized that incubation temperature induces changes in thyroid hormone and corticosterone regulation, leading to changes in expression of hormone-sensitive genes in the brain. To test this, we incubated wood duck (Aix sponsa) eggs at three different temperatures within the natural range (35.0, 35.8, and 37.0°C). We measured mRNA expression of thyroid hormone-related neuroendocrine endpoints (deiodinase 2/3, thyroid hormone receptor α/β, neural regeneration related protein, and Krueppel-like factor 9) in newly hatched ducklings and corticosterone-related neuroendocrine endpoints (mineralocorticoid receptor, glucocorticoid receptor, cholecystokinin, and brain-derived neurotrophic factor) in 15 day-old ducklings using qPCR on brain tissue from the hippocampus and hypothalamus. Contrary to our predictions, we found that mRNA expression of thyroid hormone-related endpoints in both brain areas were largely unaffected by incubation temperature, although there was a trend for an inverse relationship between mRNA expression and incubation temperature for several genes in the hypothalamus. We also found that mineralocorticoid receptor mRNA expression in the hypothalamus was lower in ducklings incubated at the low relative to the high temperatures. This study is the first to evaluate the effects of incubation temperature on mRNA expression of neuroendocrine endpoints in the developing avian brain and suggests that these particular endpoints may be largely resistant to changes in incubation temperature. Thus, further research into the proximate mechanisms for incubation temperature-induced developmental plasticity is needed.

Leptin‐induced Endothelial Dysfunction is Mediated by Endothelial Mineralocorticoid Receptor Activation in Premenopausal and Pregnant Females

Obesity is the most significant risk factor for diabetes and cardiovascular diseases. Clinical evidence indicates that mineralocorticoid receptor (MR) antagonists are more efficacious for cardiovascular treatment in obese and diabetic females than in males, however, the origin of this sex‐specific effect is unknown. Our lab previously showed that adipocyte‐derived leptin is required for endothelial dysfunction (EnDn) in obese diabetic female mice, which develops via leptin‐induced aldosterone secretion and subsequent MR activation. We further showed that female mice are more sensitive to aldosterone‐induced EnDn than males via an as yet unknown endothelial factor. We hypothesized that females express higher endothelial MR (ECMR) expression than males which predisposes females to obesity‐associated EnDn. RT‐PCR analysis in isolated aortic endothelial cells of Balb/C lean mice revealed a higher MR mRNA expression in females compared to males (2.9±0.5‐fold, P&lt;0.05), however, no such difference was observed in non‐endothelial cells (0.1±0.3‐fold). Similarly, human endothelial cells from female patients exhibited higher ECMR mRNA (2.0±0.2 fold, P&lt;0.05) and protein expression in endothelial cells (0.35±0.1 male vs 1.15±0.2 female ratio/actin, P&lt;0.05) compared to those from males. Obesity and hyperglycemia in Agouti female mice was associated with a further increase in ECMR mRNA expression (2.65±0.4 fold from wild‐type female, P&lt;0.05). Female sex‐hormone suppression (ovariectomy) decreased ECMR mRNA expression in female mice (−0.8±0.2−fold from sham, P&lt;0.05), which was restored by progesterone treatment (−0.1±0.1‐fold from sham). Progesterone dose‐dependently increased ECMR protein expression in human endothelial cells in vitro (0.1–100 ng/ml range, P&lt;0.05), indicating that progesterone induces ECMR. To assess a potential role for ECMR in leptin‐mediated EnDn we measured aorta relaxation responses to acetylcholine ex vivo (10−9–10−5M concentration). Leptin infusion (0.9mg/kg/day, 7 days) induced EnDn in intact ECMR female mice (P&lt;0.05), however, specific deletion of ECMR protected female mice from leptin‐induced EnDn indicating that leptin‐mediated EnDn is mediated by ECMR activation in females. We further discovered that ECMR mRNA increased with circulating progesterone levels in pregnant mice (9.2±0.2‐fold from nonpregnant, P&lt;0.05), indicating a potential role for ECMR in obesity‐associated cardiovascular diseases of pregnancy. Our preliminary data indicate that leptin infusion impairs EnDn in pregnant mice both compared to virgin leptin‐infused female and normal pregnant mice (P&lt;0.05). Collectively, these data indicate that progesterone increases ECMR expression in female mice which predisposes obese and pregnant female mice to leptin‐induced EnDn. Therefore, MR antagonists may be more efficacious for cardiovascular treatment in obese premenopausal women via ECMR inhibition.Support or Funding InformationNIH 1R01HL130301‐01, AHA 16IRG27770047, AHA 17POST33660678, NIH 5F32HL136191‐02.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Migratory state is not associated with differences in neural glucocorticoid or mineralocorticoid receptor expression in pine siskins

Abstract Although the endocrine system likely plays an important role in orchestrating the transition to a migratory state, the specific mechanisms by which this occurs remain poorly understood. Changes in glucocorticoid signaling are one proposed mechanism that may be important in migratory transitions. Although previous work has focused on the role of changes in circulating glucocorticoids, another potential mechanism is changes in the expression of its cognate receptors. Here, we test this hypothesis by comparing mRNA expression of the genes for the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in two brain regions implicated in the regulation of migratory behavior (the hippocampus and hypothalamus) in pine siskins (Spinus pinus) sampled before or after the transition to a spring nomadic migratory state. Compared to pre-migratory birds, migratory birds had body conditions more indicative of physiological preparations for migration (e.g., larger body mass), and greater levels of nocturnal migratory restlessness. However, we found no differences between pre-migratory and migratory birds in the expression of GR or MR mRNA in either the hippocampus or hypothalamus. Thus, differences in expression of receptors for glucocorticoids do not appear to underly the observed differences in physiology and behavior across a migratory transition. Taken together with previous results showing no change in circulating corticosterone levels during this transition, our findings provide no evidence for a role of glucocorticoid signaling in the spring migratory transition of this species.

Role of corticosterone in anxiety- and depressive-like behavior and HPA regulation following prenatal alcohol exposure

Prenatal alcohol exposure (PAE) is known to cause dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, including hyperresponsivity to stressors. Dysregulation of the HPA axis plays a role in vulnerability to stress-related disorders, such as anxiety and depression. Thus, the effects of PAE on HPA function may result in increased vulnerability to the effects of stress and, in turn, lead to the development of stress-related disorders. Indeed, individuals prenatally exposed to alcohol have an increased risk of developing anxiety and depression. However, it is unclear whether hypersecretion of corticosterone (CORT) in response to stress per se is involved with mediating differential effects of stress in PAE and control animals. To investigate the role of CORT in mediating effects of stress in both adult females and males following PAE, adrenalectomy with CORT replacement (ADXR) was utilized to produce similar CORT levels among prenatal treatment groups before exposure to chronic unpredictable stress (CUS). Anxiety-like behavior was evaluated using the open field and elevated plus maze, and depressive-like behavior was examined in the forced swim test. Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA expression was assessed in the medial prefrontal cortex (mPFC), amygdala, and hippocampal formation. Under the non-CUS condition, PAE alone differentially altered anxiety-like behavior in sham but not ADXR females and males, with females showing decreased anxiety-like behavior but males exhibiting increased anxiety-like behavior compared to their control counterparts. There were no effects of PAE alone on depressive-like in females or males. PAE also decreased GR mRNA expression in the hippocampal formation in females but had no effects on MR or GR mRNA expression in any brain region in males. CUS had differential effects on anxiety- and depressive-like behavior in PAE and control animals, and these effects were sex dependent. Importantly, ADXR unmasked differences between PAE and control animals, demonstrating that CORT may play a differential role in modulating behavior and HPA activity/regulation in PAE and control animals, and may do so in a sex-dependent manner.

Seasonally sympatric but allochronic: differential expression of hypothalamic genes in a songbird during gonadal development.

Allochrony, the mismatch of reproductive schedules, is one mechanism that can mediate sympatric speciation and diversification. In songbirds, the transition into breeding condition and gonadal growth is regulated by the hypothalamic-pituitary-gonadal (HPG) axis at multiple levels. We investigated whether the difference in reproductive timing between two seasonally sympatric subspecies of dark-eyed juncos (Junco hyemalis) was related to gene expression along the HPG axis. During the sympatric pre-breeding stage, we measured hypothalamic and testicular mRNA expression of candidate genes via qPCR in captive male juncos. For hypothalamic mRNA, we found our earlier breeding subspecies had increased expression of gonadotropin-releasing hormone (GnRH) and decreased expression of androgen receptor, oestrogen receptor alpha and mineralocorticoid receptor (MR). Subspecies did not differ in expression of hypothalamic gonadotropin-inhibitory hormone (GnIH) and glucocorticoid receptor (GR). While our earlier breeding subspecies had higher mRNA expression of testicular GR, subspecies did not differ in testicular luteinizing hormone receptor, follicle-stimulating hormone receptor or MR mRNA expression levels. Our findings indicate increased GnRH production and decreased hypothalamic sensitivity to sex steroid negative feedback as factors promoting differences in the timing of gonadal recrudescence between recently diverged populations. Differential gene expression along the HPG axis may facilitate species diversification under seasonal sympatry.

Rapid acclimation of the cortisol stress response in adult turquoise killifish Nothobranchius furzeri.

The turquoise killifish Nothobranchius furzeri is an increasingly popular model species for comparative vertebrate research, and the basic physiology including responses to stressful stimuli are of primary interest. We exposed adult killifish to a single or repeated periods of acute confinement followed by analysis of tissue cortisol and plasma cortisol concentrations. Individuals were also sampled for messenger RNA (mRNA) expression of corticotropin-releasing hormone (CRH), mineralocorticoid receptor (MR), and glucocorticoid receptor (GR) in the brain to examine the effects of repeated stress events on constitutive expression of these important stress axis components. Following a single 30-minute confinement stress, male plasma cortisol significantly differed from baseline (p = 0.04). Both male and female whole-body cortisol were significantly increased (p = 0.004 and p = 0.04, respectively) at 15 and 30 minutes poststress. Despite obvious dimorphic behavior and morphology, cortisol concentrations did not differ between the sexes. Exposure to daily repeated confinement for one week altered the cortisol response in both sexes. Time 0, 15, and 60 minutes poststress cortisol concentrations were depressed in repeatedly stressed males (p ≤ 0.05), and times 0, 30 and 120 minutes poststress cortisol concentrations were depressed in repeatedly stressed females (p ≤ 0.05). Constitutive expression of CRH, MR, and GR mRNA in the brain following one week of repeated stress events did not differ among treatments or sexes. This study introduces the first description of hypothalamic-pituitary-interrenal axis activity in this important model species. Reduced cortisol production in repeatedly stressed adult killifish suggests acclimation to repeated stressors. Furthermore, acclimation was rapid, and plasma cortisol concentrations altered significantly in as little as one week.

Sex differences in hippocampal mineralocorticoid and glucocorticoid receptor mRNA expression in response to acute mate pair separation in zebra finches (Taeniopygia guttata).

Mate separation has been shown to mediate changes in physiological and behavioral processes via activation of the hypothalamo-pituitary-adrenal (HPA) axis in both mammalian and avian species. To elucidate the neural mechanisms associated with changes in the HPA axis in response to social stress, we investigated the effects of mate pair separation on circulating corticosterone concentrations as well as gene expression levels of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and corticotropin releasing hormone (CRH) in the hypothalamus and hippocampus of both male and female zebra finches, a species that forms strong pair bonds. Zebra finches (Taeniopygia guttata) were housed three to a cage (a mated pair plus a stimulus female), and were assigned to one of three new housing treatment groups: (1) male or female removed from their respective mate and placed in a cage with a new opposite sex conspecific and stimulus female (2) male or female that remained with their mate, but a new stimulus female was introduced, or (3) the subjects were handled but not separated from their mate or the stimulus female. After 48 hr in the new housing condition, we observed significant increases in plasma corticosterone concentrations in response to both mate pair and stimulus female separation. No significant differences in MR, GR, or CRH mRNA expression in the hypothalamus were observed in response to any treatment for both males and females. Females exhibited a significant up regulation in hippocampal MR, but not GR mRNA, whereas males exhibited a significant down regulation of both hippocampal MR and GR mRNA in response to mate pair separation. Thus, the hippocampus appears to play a key role in regulating sex specific responses to social stressors.

Female Balb/C Mice Develop Salt‐sensitive Hypertension and Endothelial Dysfunction in Association with Activation of the Renin‐Angiotensin Aldosterone System

Clinical data suggest that salt‐sensitive hypertension is more prominent in woman than men. In contrast, current rodent models of salt‐sensitivity demonstrate more pronounced salt‐sensitive phenotypes in males than females. Therefore, the sex‐specific mechanisms via which females develop salt‐sensitive hypertension are poorly understood. Our lab has found that high salt diet (HS) induces sex‐specific increases in mean arterial pressure (BP) in female Balb/C mice. We have also previously shown that mineralocorticoid receptor (MR) activation is implicated in BP and endothelial regulation in female, but not male, mouse models of hypertension. However, whether renin‐angiotensin aldosterone system (RAAS) activation, and subsequent increase in MR activity, is implicated in female Balb/C salt‐sensitive hypertension is unknown. Therefore, we hypothesized that HS‐induced hypertension and endothelial dysfunction in female Balb/C mice is associated with increased RAAS activation and ablated by MR inhibition. To assess BP in male and female Balb/C mice, 10 week old mice were implanted with radiotelemeters (carotid artery) for continuous recording of BP during baseline (normal 0.2% salt (NS) diet, 7 days) and HS feeding (0.4% NaCl, 7 days). BP increased in female mice in response to HS (104.6±3.5 NS vs 114.0±2.8 HS mmHg, P&lt;0.05, n=5), however, no such increase was observed in male mice (102.6±4.0 NS vs 102.1±3.4 HS mmHg, n=5). Endothelial function was assessed as vascular relaxation to acetylcholine (10−9M–10−5M, nonlinear fit curve). HS female mice demonstrated impaired endothelial function compared to NS females (P&lt;0.05, n=7–8) while no such dysfunction developed in HS male mice (n=7–8). RT‐PCR analysis of renal tissues showed that mRNA expression of renin (1.4±0.1‐fold change, P&lt;0.05, n=5), angiotensinogen (4.4±0.2‐fold change, P&lt;0.05, n=5), MR (1.6±0.2‐fold change, P&lt;0.05, n=5) and epithelial sodium channel‐α (1.3±0.1‐fold change, P&lt;0.05, n=5) were increased by HS in female mice compared to NS females, indicating an activation of RAAS by HS in Balb/C females. Of the RAAS mRNA expressions assessed, only renal MR mRNA expression was increased in male mice on HS compared to NS males (1.30±0.1‐fold change, P&lt;0.05, n=5). Importantly, plasma aldosterone levels were decreased in male HS mice compared to NS males (490±135 NS vs 160±35 HS pg/ml, P&lt;0.05, n=8–16), however, HS did not decrease aldosterone levels in female mice compared to NS females (562±125 NS vs 334±64 HS pg/ml, n=9–15), demonstrating a sex‐specific preservation of aldosterone levels in the presence of HS in female Balb/C mice. Furthermore, BP increases and endothelial dysfunction in HS female mice were ablated in response to the MR antagonist eplerenone (200 ng/kg/day, drinking water) (96.1±7.1 mmHg, P&lt;0.05, n=4), whereas no effects were observed in male HS eplerenone mice (106.1±5.8 mmHg, n=4). These data indicate that female Balb/C mice develop sex‐specific hypertension and endothelial dysfunction in association with RAAS activation which is dependent on MR activation. Therefore, the female Balb/C mouse may be an important model for studies of the mechanisms of salt‐sensitive hypertension in women.Support or Funding InformationThis work was supported by the following: NIH 1R01HL130301‐01, AHA 16IRG27770047, NIH 5F32HL136191‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

11β-HSD Types 1 and 2 in the Songbird Brain.

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