Glucocorticoid Receptor Content Research Articles

Are receptor concentrations correlated across tissues within individuals? A case study examining glucocorticoid and mineralocorticoid receptor binding.

Hormone receptors are a necessary (although not sufficient) part of the process through which hormones like corticosterone create physiological responses. However, it is currently unknown to what extent receptor concentrations across different target tissues may be correlated within individual animals. In this study, we examined this question using a large dataset of radioligand binding data for glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in 13 different tissues in the house sparrow (Passer domesticus) (n=72). Our data revealed that individual house sparrows tended to exhibit higher or lower receptor binding across all tissues, which could be part of what creates the physiological and behavioral syndromes associated with different hormonal profiles. However, although statistically significant, the correlations between tissues were very weak. Thus, when each tissue was independently regressed on receptor concentrations in the other tissues, multivariate analysis revealed significant relationships only for sc fat (for GR) and whole brain, hippocampus, kidney, omental fat, and sc fat (for MR). We also found significant pairwise correlations only between receptor concentrations in brain and hippocampus, and brain and kidney (both for MR). This research reveals that although there are generalized individual consistencies in GR and MR concentrations, possibly due to such factors as hormonal regulation and genetic effects, the ability of 2 different tissues to respond to the same hormonal signal appears to be affected by additional factors that remain to be identified.

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α1- and β2-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [3H]-prazosin and [3H]-CGP-12177 for determination of α1- and β2-AR and [3H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α1-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α1-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas β2-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α1-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

The selective glucocorticoid receptor modulator CORT108297 restores faulty hippocampal parameters in Wobbler and corticosterone-treated mice

Mutant Wobbler mice are models for human amyotrophic lateral sclerosis (ALS). In addition to spinal cord degeneration, Wobbler mice show high levels of blood corticosterone, hyperactivity of the hypothalamic–pituitary–adrenal axis and abnormalities of the hippocampus. Hypersecretion of glucocorticoids increase hippocampus vulnerability, a process linked to an enriched content of glucocorticoid receptors (GR). Hence, we studied if a selective GR antagonist (CORT108297) with null affinity for other steroid receptors restored faulty hippocampus parameters of Wobbler mice. Three months old genotyped Wobbler mice received s.c. vehicle or CORT108297 during 4 days. We compared the response of doublecortin (DCX)+ neuroblasts in the subgranular layer of the dentate gyrus (DG), NeuN+ cells in the hilus of the DG, glial fibrillary acidic protein (GFAP)+ astrocytes and the phenotype of Iba1+ microglia in CORT108297-treated and vehicle-treated Wobblers. The number of DCX+ cells in Wobblers was lower than in control mice, whereas CORT108297 restored this parameter. After CORT108297 treatment, Wobblers showed diminished astrogliosis, and changed the phenotype of Iba1+ microglia from an activated to a quiescent form. These changes occurred without alterations in the hypercorticosteronemia or the number of NeuN+ cells of the Wobblers. In a separate experiment employing control NFR/NFR mice, treatment with corticosterone for 5 days reduced DCX+ neuroblasts and induced astrocyte hypertrophy, whereas treatment with CORT108297 antagonized these effects. Normalization of neuronal progenitors, astrogliosis and microglial phenotype by CORT108297 indicates the usefulness of this antagonist to normalize hippocampus parameters of Wobbler mice. Thus, CORT108297 opens new therapeutic options for the brain abnormalities of ALS patients and hyperadrenocorticisms.

Chronic stress alters concentrations of corticosterone receptors in a tissue-specific manner in wild house sparrows (Passer domesticus).

The physiological stress response results in release of glucocorticoid hormones such as corticosterone (CORT). Whereas short-term activation of this response helps animals cope with environmental stressors, chronic activation can result in negative effects including metabolic dysregulation and reproductive failure. However, there is no consensus hormonal profile of a chronically stressed animal, suggesting that researchers may need to look beyond hormone titers to interpret the impacts of chronic stress. In this study, we brought wild house sparrows (Passer domesticus) into captivity. We then compared glucocorticoid and mineralocorticoid receptor concentrations in sparrows exposed either to a standardized chronic stress protocol (n=26) or to standard husbandry conditions (controls; n=20). We used radioligand binding assays to quantify receptors in whole brain, liver, kidneys, spleen, gonads, gastrocnemius and pectoralis muscle, omental and subcutaneous fat, and bib and back skin. In most tissues, CORT receptors did not differ between controls and stressed animals, although we found marginal increases in receptor density in kidney and testes in stressed birds at some time points. Only in pectoralis muscle was there a robust effect of chronic stress, with both receptor types higher in stressed animals. Increased pectoralis sensitivity to CORT with chronic stress may be part of the underlying mechanism for muscle wasting in animals administered exogenous CORT. Furthermore, the change in pectoralis was not paralleled by gastrocnemius receptors. This difference may help explain previous reports of a greater effect of CORT on pectoralis than on other muscle types, and indicate that birds use this muscle as a protein reserve.

The size of a melanin-based plumage ornament correlates with glucocorticoid receptor concentrations in the skin of that ornament

Glucocorticoid hormones such as corticosterone (CORT) play crucial roles in many physiological processes. CORT's actions are primarily mediated via binding to two receptors (glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs)) in different target tissues. CORT receptors can be independently regulated from circulating hormone titres, from tissue to tissue and even within different regions of the same tissue type. Increasing evidence has shown relationships between circulating CORT and melanin-based pigmentation in skin and feathers, yet to our knowledge, there have been no studies of CORT receptors in the skin of melanized ornaments. Male house sparrows (Passer domesticus) have a black, melanized bib, and evidence suggests that bib size is an important intraspecific signal. We examined the relationship between bib area and tissue sensitivity to CORT by quantifying GR and MR in bib skin and in adjacent paler-feathered belly skin (as a control tissue) at different life-history stages using radioligand binding assays. Males with larger bibs relative to their life-history stage had less GR in bib skin, but not belly skin, than males with smaller bibs. These results suggest a connection between the size of a melanin-based ornament and the underlying tissue's responsiveness to CORT.

Vaccination inhibits TLR2 transcription via suppression of GR nuclear translocation and binding to TLR2 promoter in porcine lung infected with Mycoplasma hyopneumoniae

Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (P<0.01), but only VI pigs demonstrated significantly diminished nuclear GR content. TLRs 1–10 were all expressed in lung, among which TLR2 was the most abundant and was significantly up-regulated (P<0.05) in NI pigs, but not in VI pigs. Accordingly, GR binding to the GR response element on TLR2 promoter was significantly increased (P<0.05) in NI pigs, but not in VI pigs. These results suggest that the inhibition of GR nuclear translocation and binding to the TLR2 promoter, which results in diminished TLR2 expression, is associated with the protective effect of vaccination on M. hyopneumoniae-induced lung lesions in the pig.

Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model.

Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.

Ligand-Induced Repression of the Glucocorticoid Receptor Gene Is Mediated by an NCoR1 Repression Complex Formed by Long-Range Chromatin Interactions with Intragenic Glucocorticoid Response Elements

Glucocorticoids are among the most potent and effective agents for treating inflammatory diseases and hematological cancers. However, subpopulations of patients are often resistant to steroid therapy, and determining the molecular mechanisms that contribute to glucocorticoid resistance is thus critical to addressing this clinical problem affecting patients with chronic inflammatory disorders. Since the cellular level of the glucocorticoid receptor (GR) is a critical determinant of glucocorticoid sensitivity and resistance, we investigated the molecular mechanisms mediating repression of glucocorticoid receptor gene expression. We show here that glucocorticoid-induced repression of GR gene expression is mediated by inhibition of transcription initiation. This process is orchestrated by the recruitment of agonist-bound GR to exon 6, followed by the assembly of a GR-NCoR1-histone deacetylase 3-containing repression complex at the transcriptional start site of the GR gene. A functional negative glucocorticoid response element (nGRE) in exon 6 of the GR gene and a long-range interaction occurring between this intragenic response element and the transcription start site appear to be instrumental in this repression. This autoregulatory mechanism of repression implies that the GR concentration can coordinate repression with excess ligand, regardless of the combinatorial associations of tissue-specific transcription factors. Consequently, the chronic nature of inflammatory conditions involving long-term glucocorticoid administration may lead to constitutive repression of GR gene transcription and thus to glucocorticoid resistance.

Intracellular glucocorticoid receptors in spleen, but not skin, vary seasonally in wild house sparrows (Passer domesticus)

Over the short-term and at physiological doses, acute increases in corticosterone (CORT) titres can enhance immune function. There are predictable seasonal patterns in both circulating CORT and immune function across many animal species, but whether CORT receptor density in immune tissues varies seasonally is currently unknown. Using radioligand binding assays, we examined changes in concentrations of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in spleen and skin in wild-caught house sparrows in Massachusetts during six different life-history stages: moult, early winter, late winter, pre-egg-laying, breeding and late breeding. Splenic GR and MR binding were highest during the pre-laying period. This may help animals respond to immune threats through increased lymphocyte proliferation and/or an increase in delayed-type hypersensitivity reactions, both of which CORT can stimulate and in which spleen is involved. A decrease in splenic GR and MR during the late breeding period coincides with low baseline and stress-induced CORT, suggesting immune function in spleen may be relatively CORT-independent during this period. We saw no seasonal patterns in GR or MR in skin, suggesting skin's response to CORT is modulated primarily via changes in circulating CORT titres and/or via local production of CORT in response to wounding and other noxious stimuli.

Early environments, glucocorticoid receptors, and behavioral epigenetics.

In 1985, a brief report published in Behavioral Neuroscience established the link between neonatal handling and concentrations of hippocampal glucocorticoid receptors (GR) in the adult rat, suggesting a neurobiological basis for the attenuated stress reactivity observed in handled versus nonhandled offspring. To celebrate the 30th anniversary of Behavioral Neuroscience, this article explores the research that preceded and followed from this brief but significant publication. Changes in hippocampal GR induced by handling were determined to be the outcome of a cascade of cellular and molecular events involving thyroid hormones, serotonin turnover, and transcription factor binding to the Nr3c1 gene, leading to increased GR mRNA and protein. Though many hypotheses were proposed for the "handling effect," the role of handling-induced changes in maternal care, particularly pup licking/grooming (LG), generated a productive scientific framework for understanding the handling phenomenon. Indeed, LG has since been demonstrated to alter GR levels through the signaling pathways described for handling. Moreover, epigenetic mechanisms have been discovered to play a critical role in the effects of early life experience and particularly in the regulation of Nr3c1. Overall, the research avenues that have evolved from the initial finding of handling-induced changes in GR have broad applications to our understanding of plasticity, resilience, and the transmission of traits across generations.

Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 °C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress.

Glucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution

Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10−6M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10−10–10−9M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18–27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.

Glucocorticoid Receptor Density Correlates with Health Risk Factors and Insulin Resistance in Caucasian and African American Subjects

Activation of the hypothalamic-pituitary-adrenal axis leads to secretion of cortisol, which binds to peripheral glucocorticoid receptor and mediates a complex series of metabolic and immune effects. Cortisol also binds to receptors in the hypothalamus and pituitary, and inhibits further secretion of adrenocorticotropic hormone thus preventing an excessive response. Excess glucocorticoid effect is seen in Cushings disease, adrenal adenomas/carcinomas and in glucocorticoid resistance. Within such pathology there are health consequences of excessive glucocorticoid action, including obesity, hypertension, and glucose intolerance or diabetes. We hypothesized that increased glucocorticoid receptor in peripheral tissue might mediate an excess glucocorticoid effect in the absence of increased cortisol secretion. The objective of the study was to investigate the relationship between glucocorticoid receptor density in leukocytes and health risk indices relevant to obesity and diabetes in a sample of Caucasian and African American subjects. Comparison of glucocorticoid receptor concentration with subject body mass index, percentage body fat, waist circumference, insulin resistance, plasma cortisol levels, gender, and lipid profiles were conducted. Increased glucocorticoid receptor density significantly correlated with body mass index, percentage body fat, waist circumference, and insulin resistance. No significant correlation was observed for glucocorticoid receptor density with lipid profiles. Furthermore, no significant differences were observed in glucocorticoid receptor density between Caucasian and African American subjects or male and female participants. Our results show that high risk health conditions, such as obesity and type-2 diabetes, may be associated with a form of hypothalamic-pituitary-adrenal axis dysfunction, characterized by localized leukocyte glucocorticoid receptor over-expression.

Detection of the acute effects of hydrocortisone in the hippocampus using pharmacological fMRI

Impaired hippocampal function is believed to be important in the pathogenesis of depression. The hippocampus contains a high concentration of both mineralocorticoid (MR) and glucocorticoid receptors (GR), and the experimental administration of corticosteroids has been reported to mimic memory impairments seen in depression. Using pharmacological functional magnetic resonance imaging (phMRI) we investigated whether hippocampal function is altered after acute administration of hydrocortisone. Changes in BOLD signal following infusion of 100mg hydrocortisone given as a rapid intravenous bolus were measured in 14 healthy volunteers in a within-subject placebo-controlled crossover design. Subsequently, subjects completed an n-back task during an fMRI scan. Hydrocortisone infusion caused a significant, time-dependent increase in fMRI BOLD signal in hippocampus reaching a maximal effect at 11–19min. The n-back task increased BOLD signal in prefrontal and parietal cortical areas and decreased it in the hippocampus. After hydrocortisone the left hippocampal decrease in BOLD signal was attenuated with the magnitude of attenuation correlating with the increase seen after hydrocortisone infusion. No difference in behavioural task performance was observed. The results suggest acute hydrocortisone has rapid direct and modulatory influences on hippocampal function, probably acting through non-genomic GR or MR signalling. Hydrocortisone infusion phMRI may be a useful tool to investigate hippocampal corticosteroid receptor function in depression.

FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer

The forkhead transcription factor FoxA1 participates in many gene regulatory events with steroid hormone receptors, one example being the integrated mouse mammary tumor virus (MMTV) promoter. Its enhancer harbors several FoxA1 binding sites. FoxA1 promotes glucocorticoid receptor (GR)-DNA binding and transcription. Here we analyze the regulatory capacity of GR, FoxA1 and hormone in quantitative terms when reconstituted with the MMTV enhancer in Xenopus oocytes. By titrating each component we demonstrate that FoxA1 is required for hormone induction at low GR concentration and that FoxA1 is a potent enhancer of GR-induced transcription. Conversely, specific DNA binding of FoxA1 at low intranuclear concentration is highly responsive to minute levels of hormone-activated GR while increased FoxA1 concentration results in constitutive binding. When bound to DNA, FoxA1 induces DNase I hypersensitivity, this is accompanied by increased acetylation, specifically at histone H4K16. Expression of FoxA1 deletion mutants demonstrated its DNA binding domain to be sufficient for DNA binding in vivo. The C-terminal and N-terminal domains both contribute to chromatin remodeling while the latter is more important for GR mediated transcription. Thus FoxA1 is primarily responsible for the chromatin presetting while GR supports chromatin presetting at low hormone concentration and transcriptional induction at high hormone concentration.

Ligand Binding Domain Mutations of the Glucocorticoid Receptor Selectively Modify the Effects with, but Not Binding of, Cofactors

We previously reported that several point mutations in the ligand binding domain (LBD) of glucocorticoid receptors (GRs) marginally affect the binding affinity of the synthetic glucocorticoids dexamethasone (Dex) and deacylcortivazol (DAC). However, these mutations dramatically alter the efficacy (A(max)) and potency (EC(50)) of agonists, along with the partial agonist activity (PAA) of the antisteroid Dex-mesylate (DM), for gene induction and repression in a steroid-dependent manner. This was proposed to result, in part, from altered protein-protein interactions in the complex of GR with the coactivator TIF2 despite normal TIF2 binding. To explore the generality of this phenomenon, we now ask whether these mutations also affect the transactivation properties, but not binding, of other GR-bound factors. We find that an elevated concentration of GR, to probe unidentified cofactors, or of the comodulator Ubc9 does not reverse the effects of GR LBD mutations that increase the EC(50) and lower the PAA with the GREtkLUC reporter in both CV-1 and U2OS cells. This behavior is more dramatic with Ubc9 and the isolated GR LBD fused to the GAL4 DNA binding domain, despite normal binding of Ubc9 to the mutant GRs. Similar effects, albeit gene, steroid, and transcriptional property-specific, are seen with full-length GRs and three endogenous genes in U2OS cells. Thus, changes in simple steady-state binding capacities of mutant receptors for factors cannot account for the modified transcriptional properties. In all cases, the nuclear translocation of Dex- and DAC-bound wild-type and mutant receptors is the same. These results are consistent with the earlier results with TIF2 and support the hypothesis that small changes in the GR LBD can alter the activities of the bound cofactor without modifying cofactor binding. We propose that this separation of binding and the modulation of transactivation parameters occurs for a wide variety of GR-associated cofactors.

Hyperactivity of the hypothalamus–pituitary–adrenal axis in lipopolysaccharide-induced neurodevelopmental model of schizophrenia in rats: Effects of antipsychotic drugs

Recent data indicate that a significant number of schizophrenic patients are hypercortisolemic and that glucocorticoids are involved in the pathogenesis of schizophrenia. The aim of the present study was to evaluate whether behavioural schizophrenia-like changes in the lipopolysaccharide (LPS)-induced neurodevelopmental model of this brain disorder are associated with alterations in the level of plasma corticosterone, the concentration of glucocorticoid receptors and the amount of the immunophilin FKBP51, the glucocorticoid receptor co-chaperone, in the hippocampus and frontal cortex. We found that the adult offspring of prenatally LPS-treated rats showed a deficit in prepulse inhibition (PPI), an enhancement of amphetamine-induced locomotor activity, an elevated plasma level of corticosterone and a decrease in both the glucocorticoid receptor level in the hippocampus and the FKBP51 concentration in the frontal cortex. Most of these changes were reversed by the atypical antipsychotic drug clozapine, whereas chlorpromazine had no effect on PPI but attenuated the amphetamine-induced hyperactivity and normalised the hippocampal level of glucocorticoid receptors. The changes in the level of plasma corticosterone and cortical FKBP51 were attenuated by chlorpromazine in female offspring only. This study supports the hypothesis of hypothalamic–pituitary–adrenal (HPA) axis hyperactivity in schizophrenia and suggests that this hyperactivity results from a decrease in the hippocampal glucocorticoid receptor level and a decrease in FKBP51 in the frontal cortex.

Effects of housing and short-term transportation on hormone and lymphocyte receptor concentrations in beef cattle

The experiment was designed to evaluate the effects of housing system and short-term transportation on the pituitary and adrenal response and on blood progesterone concentrations of beef cattle. Since the use of steroid hormones in farm animals has been banned in the EU (Council Directive 96/22/EC), it seems important to study the possible modifications in serum progesterone concentrations induced by stress in cattle. Thirty-two, 6 months old male Piedmontese beef cattle (16 reared in a littered loose house, Group A, and 16 housed in a littered tying stall barn, Group B) were blood sampled at T1 (6 months old), T2 (12 months old), T3 (18 months old, before transportation to the slaughterhouse) and T4 (after transportation to the slaughterhouse) in order to measure hormonal concentrations and lymphocyte glucocorticoid (GR) and β-adrenergic (β-AR) receptor concentrations. Circulating hormone concentrations were measured using commercial radioimmunoassay kits, whereas lymphocyte receptor density was determined through binding assays. In beef cattle housed in tie stall barn a significant increase in serum cortisol concentration was observed at T3, whereas there was no effect of the housing system on blood progesterone concentrations. Short-term transportation caused a significant increase in blood cortisol and catecholamine concentrations in both groups, whereas lymphocyte GR and β-AR significantly decreased in Group A. Our data confirm the activation of the hypothalamic–pituitary–adrenal axis and the catecholaminergic system in short-term transportation and suggest that the stress-induced increase in circulating progesterone concentrations does not exceed the limit established by pending legislation.

Regular exercise prevents the development of hyperglucocorticoidemia via adaptations in the brain and adrenal glands in male Zucker diabetic fatty rats

We determined the effects of voluntary wheel running on the hypothalamic-pituitary-adrenal (HPA) axis, and the peripheral determinants of glucocorticoids action, in male Zucker diabetic fatty (ZDF) rats. Six-week-old euglycemic ZDF rats were divided into Basal, Sedentary, and Exercise groups (n = 8-9 per group). Basal animals were immediately killed, whereas Sedentary and Exercising rats were monitored for 10 wk. Basal (i.e., approximately 0900 AM in the resting state) glucocorticoid levels increased 2.3-fold by week 3 in Sedentary rats where they remained elevated for the duration of the study. After an initial elevation in basal glucocorticoid levels at week 1, Exercise rats maintained low glucocorticoid levels from week 3 through week 10. Hyperglycemia was evident in Sedentary animals by week 7, whereas Exercising animals maintained euglycemia throughout. At the time of death, the Sedentary group had approximately 40% lower glucocorticoid receptor (GR) content in the hippocampus, compared with the Basal and Exercise groups (P < 0.05), suggesting that the former group had impaired negative feedback regulation of the HPA axis. Both Sedentary and Exercise groups had elevated ACTH compared with Basal rats, indicating that central drive of the axis was similar between groups. However, Sedentary, but not Exercise, animals had elevated adrenal ACTH receptor and steroidogenic acute regulatory protein content compared with the Basal animals, suggesting that regular exercise protects against elevations in glucocorticoids by a downregulation of adrenal sensitivity to ACTH. GR and 11beta-hydroxysteroid dehydrogenase type 1 content in skeletal muscle and liver were similar between groups, however, GR content in adipose tissue was elevated in the Sedentary groups compared with the Basal and Exercise (P < 0.05) groups. Thus, the gradual elevations in glucocorticoid levels associated with the development of insulin resistance in male ZDF rats can be prevented with regular exercise, likely because of adaptations that occur primarily in the adrenal glands.

Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae

The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in Saccharomyces cerevisiae. In this report we demonstrate that the in vivo GR phosphorylation pattern, hormone dependency and interdependency of phosphorylation events were similar in yeast and mammalian cells. GR phosphorylation at S246 exhibited inhibitory effect on S224 and S232 phosphorylation, suggesting the conservation of molecular mechanisms that control this interdependence between yeast and mammalian cells.To assess the effects of GR phosphorylation the mutated GR derivatives T171A, S224A, S232A, S246A were overexpressed and their transcriptional activity was analysed. These receptor derivatives displayed significant hormone inducible transcription when overexpressed in S. cerevisiae. We have established an inducible methionine expression system, which allows the close regulation of the receptor protein levels to analyse the dependence of GR function on its phosphorylation and protein abundance. Using this system we observed that GR S246A mutation increased its activity across all of the GR concentrations tested. The activity of the S224A and S246A mutants was mostly independent of GR protein levels, whereas the WT, T171A and S232A mediated transcription diminished with declining GR protein levels. Our results suggest that GR phosphorylation at specific residues affects its transcriptional functions in a site selective manner and these effects were directly linked to GR dosage.