Corticosterone Output Research Articles

Vitamin D3 application and factors of its activity in the adrenal cortex of andropausal rats: A functionally-histological study

Introduction and aimVitamin D supplementation in aging subjects manifests a positive effect on various health-related parameters. We performed a functionally-histological analysis of the adrenal cortex regarding the factors of vitamin D activity and corticosterone output after vitamin D3 application in a rat model of the andropause. Material and methodsMiddle-aged Wistar rats were divided into sham operated (SO; n=8), orchidectomized (Orx; n=8) and vitamin D3-treated orchidectomized (Orx+vit. D; n=8) groups. Vitamin D3 (5 μg/kg b.m.) was administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using histochemistry/immunohistochemistry, stereology, ultrastructural and biochemical analyses. ResultsOrchidectomy (Orx) decreased the adrenal cortex-related volume densities of vascular (p<0,0001), vitamin D receptor (VDR; p<0,0166), cytochrome P450 oxidase 2R1 (CYP 2R1; p<0,0001) and cytochrome P450 oxidase 24 (CYP 24; p<0,0001) depots, but increased the volume density of cytochrome P450 27B1 (CYP 27B1; p<0,0001) depots. In Orx+vit. D rats, increase of the adrenal cortex-related volume densities of collagen (p<0,0001), VDR (p<0,0001) and CYP 2R1 (p<0,0001) depots as well as the lipid-droplet diameter (p<0,0001) in adrenocortical outer zona fasciculata cells was observed, while a decrease of volume densities of the vascular (p<0,0001), CYP 27B1 (p<0,0001) and CYP 24 (p<0,0001) depots was registered, all versus Orx group. Plasma level of ACTH was decreased (p=0,0155) and serum concentrations of 25-hydroxyvitamin D3 and corticosterone were increased (p<0,0001 and p=0,0187, respectively), all after the same treatment. ConclusionsIncreased corticosterone output after vitamin D3 application to andropausal rats appears not to be related to increased availability of 25-hydroxyvitamin D3 and decreased degradation of 1,25-dihydroxyvitamin D3 in adrenal tissue, but rather involves the central regulatory mechanisms.

The Effects of Long-Term Exposure to Moderate Heat on Rat Pituitary ACTH Cells: Histological and Hormonal Study

Abstract Global warming causes an increased ambient temperature and prolonged heatwaves during the summer, which represent stressogenic factors affecting the hypothalamo-pituitary-adrenocortical (HPA) axis in mammals. The aim of this study was to investigate the effects of long-term (7-60 days) exposure to moderately elevated ambient temperature (35 ± 1°C) on the histological aspect and secretory ability of pituitary adrenocorticotropic (ACTH) cells, as well as on the corticosterone output, in adult rats. Stereological parameters of ACTH cells were estimated upon immunohistochemistry. The blood concentrations of ACTH and corticosterone were determined by immunoassays. The volume of ACTH cells in rats exposed to moderately high temperature for 7, 14, 21, 30 and 60 days decreased (p&lt;0.05) by 18.1%, 14.5%, 13.5%, 8.6% and 14.2% respectively, compared to the same parameter in the controls. The volume density of ACTH cells in the groups exposed to elevated temperature for 7, 14, 21, 30 and 60 days decreased (p&lt;0.05) by 40.0%, 33.3%, 26.7%, 13.3% and 26.7% respectively, in comparison with control rats. The plasma concentration of ACTH varied differently (p&lt;0.05) with the duration of exposure to the elevated temperature. The serum concentration of corticosterone was decreased (p&lt;0.05) by 54.9%, 24.4%, 29.9%, 21.1% and 24.4% in groups subjected to moderately high temperature for 7, 14, 21, 30 and 60 days respectively, all compared to the control value. Despite some signs of functional recovery of ACTH cells during the treatment, the impression is that the long-term character of this stressor overcomes the capacity of the HPA axis for resistance.

Hindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat

Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen- and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERβ) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERβ mediates local glycogen breakdown. DVC ERs also regulate VMN neurotransmitter signaling of energy sufficiency [γ-aminobutyric acid] or deficiency [nitric oxide, steroidogenic factor-1] during IIH. Neither hindbrain ER mediates IIH-associated diminution of VMN norepinephrine (NE) content. Both ERs oppose hypoglycemic hyperglucagonemia, while ERβ contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ERs control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -β implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.

Nicotinamide phosphoribosyltransferase and the hypothalamic-pituitary-adrenal axis of the rat.

Nicotinamide phosphoribosyltransferase (Nampt), also termed visfatin, catalyses the rate‑limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. In addition to its intracellular function (iNampt), extracellular Nampt (eNampt) also affects numerous intracellular signalling pathways. The current study investigated the role of Nampt in the regulation of the hypothalamic‑pituitary‑adrenal (HPA) axis in rats. At 1h after intraperitoneal administration of eNampt (4µg/100g) in adult male rats, serum adrenocorticotropic hormone(ACTH) and aldosterone levels remained unchanged, while corticosterone levels were notably elevated compared with the control group, as determined by ELISA. The results of reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) demonstrated that, in the hypothalami of eNampt‑treated rats, the mRNA expression levels of Fos proto‑oncogene, which is also termed c‑Fos, were not significantly different compared with the control group; however, the mRNA expression levels of proopiomelanocortin (POMC) were markedly increased in the pituitary gland of eNampt‑treated rats compared with the control group. Furthermore, in hypothalamic explants, ELISA results demonstrated that the addition of the eNampt protein exhibited no effect on corticotropin‑releasing hormone (CRH) release into the incubation medium and prevented potassium ion‑induced CRH release. Additionally, the eNampt‑induced increase in ACTH output by pituitary gland explants was not statistically significant, compared with the control group. However, RT‑qPCR indicated that exposure of pituitary gland explants to eNampt and CRH increased the levels of POMC mRNA expression; the effect of eNampt, but not CRH, was inhibited by FK866, which is a specific Nampt inhibitor. In primary rat adrenocortical cell cultures, eNampt exhibited no effect on basal aldosterone or corticosterone secretion, while increases in aldosterone and corticosterone levels in response to ACTH were retained. To assess the potential role of iNampt in the regulation of adrenal steroidogenesis, experiments involving a specific Nampt inhibitor, FK866, were performed. Exposure of cultured cells to FK866 notably lowered basal aldosterone and corticosterone output compared with the control group, and completely eliminated the response of cultured cells to ACTH. The results of the present study indicated that the injected eNampt may have increased the corticosterone serum levels by acting at the pituitary level. In addition, iNampt may exert a tonic stimulating effect on the secretion of aldosterone and corticosterone from rat adrenocortical cells, as normal iNampt levels were required to retain the response of cultured rat adrenocortical cells to ACTH. Thus, these data suggest an important physiological role of both iNampt and eNampt in the regulation of the HPA axis activity in the rat.

NNT is a key regulator of adrenal redox homeostasis and steroidogenesis in male mice.

Nicotinamide nucleotide transhydrogenase, NNT, is a ubiquitous protein of the inner mitochondrial membrane with a key role in mitochondrial redox balance. NNT produces high concentrations of NADPH for detoxification of reactive oxygen species by glutathione and thioredoxin pathways. In humans, NNT dysfunction leads to an adrenal-specific disorder, glucocorticoid deficiency. Certain substrains of C57BL/6 mice contain a spontaneously occurring inactivating Nnt mutation and display glucocorticoid deficiency along with glucose intolerance and reduced insulin secretion. To understand the underlying mechanism(s) behind the glucocorticoid deficiency, we performed comprehensive RNA-seq on adrenals from wild-type (C57BL/6N), mutant (C57BL/6J) and BAC transgenic mice overexpressing Nnt (C57BL/6JBAC). The following results were obtained. Our data suggest that Nnt deletion (or overexpression) reduces adrenal steroidogenic output by decreasing the expression of crucial, mitochondrial antioxidant (Prdx3 and Txnrd2) and steroidogenic (Cyp11a1) enzymes. Pathway analysis also revealed upregulation of heat shock protein machinery and haemoglobins possibly in response to the oxidative stress initiated by NNT ablation. In conclusion, using transcriptomic profiling in adrenals from three mouse models, we showed that disturbances in adrenal redox homeostasis are mediated not only by under expression of NNT but also by its overexpression. Further, we demonstrated that both under expression or overexpression of NNT reduced corticosterone output implying a central role for it in the control of steroidogenesis. This is likely due to a reduction in the expression of a key steroidogenic enzyme, Cyp11a1, which mirrored the reduction in corticosterone output.

El ejercicio físico moderado evita la aparición de insulinorresistencia y de alteraciones morfofuncionales adrenocorticales en ratas tratadas con una dieta rica en sacarosa

Introducción: niveles elevados de glucocorticoides se asocian a las alteraciones somáticas y bioquímicas presentes en pacientes y en animales con insulinorresistencia (IR). Hemos demostrado previamente que la IR inducida por una dieta rica en sacarosa (DRS) induce cambios morfológicos y funcionales a nivel adrenocortical y que estas alteraciones pueden evitarse mediante la administración simultánea de un agonista PPAR-γ.Objetivo: evaluar el impacto de un protocolo de ejercicio moderado sobre las alteraciones morfológicas y funcionales adrenocorticales asociadas con el desarrollo de IR inducida por una DRS administrada durante siete semanas.Metodología: Resultados: los animales (ratas Wistar macho adultas) tratados con la DRS (agregado de sacarosa al 30% en el agua de bebida) mostraron un incremento del peso corporal y de los panículos adiposos, así como de los niveles séricos de glucosa, insulina y triglicéridos. La respuesta glucémica a la administración de insulina i.p. se vio claramente menoscabada. Se observó una infiltración lipídica de la corteza adrenal, con aumento de la expresión de proteínas esteroidogénicas y marcadores de inflamación (IL-1β, TNF-α, iNOS, COX-2) y un incremento marcado de la corticosteronemia basal. El protocolo de ejercicio consistió en correr en una cinta continua adaptada especialmente durante un máximo de 7 min/día. Este ejercicio moderado previno la aparición de los cambios somáticos y bioquímicos característicos del estado de IR y la infiltración lipídica adrenocortical, revirtiendo además los cambios inflamatorios y normalizando la corticosteronemia.Conclusiones: nuestros resultados subrayan el rol deletéreo del consumo exagerado de carbohidratos simples conteniendo fructosa y sugieren que el ejercicio moderado podría tener efectos adicionales cuando se emplea en el tratamiento de la IR.

HIV-1 proteins accelerate HPA axis habituation in female rats

Congenital infection by the Human Immunodeficiency Virus (HIV) has been shown to lead to multiple co-morbidities, and people living with HIV have a higher incidence of affective and anxiety disorders. A marked increase in mood disorders is evident during the sensitive phase of adolescence and this is further pronounced in females. Depression has been linked to dysfunction of the intracellular response system to corticosteroids at the level of the hippocampus (HC) and prefrontal cortex (PFC) with a notable role of the glucocorticoid receptor (GR) and its co-chaperones (FKBP5 and FKBP4). The current study examined the extent to which HIV protein expression in adolescent female rats altered the stress response at both the level of corticosterone output and molecular regulation of the glucocorticoid receptor in the brain. WT and HIV-1 genotype female rats were randomly allocated in control, acute stress and repeat stress groups. Corticosterone plasma levels and expression of GR, FKBP4, and FKBP5 in the HC and PFC were measured. The presence of HIV-1 proteins facilitates habituation of the corticosterone response to repeated stressors, such that HIV-1 TG rats habituated to repeated restraint and WT rats did not. This was reflected by interactions between stress exposure and HIV-1 protein expression at the level of GR co-chaperones. Although expression of the GR was similarly reduced after acute and repeat stress in both genotypes, expression of FKBP5 and FKBP4 was altered in a brain-region specific manner depending on the duration of the stress exposure and the presence or absence of HIV-1 proteins. Collectively, the data presented demonstrate that HIV-1 proteins accelerate habituation to repeated stressors and modify the influence of acute and repeat stressors on GR co-chaperones in a brain region-specific manner.

Context‐dependent stress responses and their connections to fitness in a landscape of fear

AbstractThe acute glucocorticoid stress response is presumed to facilitate escape from life‐threatening situations such as predation and thus it is assumed to be linked to fitness. However, the fitness effects of glucocorticoid reactivity remain controversial, as these effects may be context‐dependent. Individuals differing in their emphasis on current versus future reproduction may differ in their risk‐taking under threat of predation; this variation in risk‐taking may be mediated by variations in stress reactivity. We set out to test whether predation risk (island‐ and year‐specific proportion of depredated nests) modified the relationships between stress responsiveness and current reproductive investment (clutch weight) and between stress responsiveness and reproductive success (viable proportion of the clutch) in the long‐lived female eider Somateria mollissima. This study system shows large spatial and annual fluctuations in predation risk, indexed by the annual island‐specific proportion of depredated nests. The capture stress‐related corticosterone output was attenuated with increasing clutch weight under low predation pressure but elevated under severe predation pressure, and females in well‐concealed nests had lower stress responsiveness. The viable proportion of the clutch decreased with increasing corticosterone reactivity under low to moderate predation pressure, but slightly increased under severe predation pressure. The acute stress response may thus mediate adaptive plasticity; dampened stress reactivity may ensure successful reproduction under low predation threat or in nest sites reducing detection by visual predators, whereas preparing for potential attacks may be favoured under elevated predation risk.

Exposure to a social stressor disrupts the community structure of the colonic mucosa-associated microbiota.

BackgroundThe microbiota of the mammalian gastrointestinal (GI) tract consists of diverse populations of commensal bacteria that interact with host physiological function. Dysregulating these populations, through exogenous means such as antibiotics or dietary changes, can have adverse consequences on the health of the host. Studies from laboratories such as ours have demonstrated that exposure to psychological stressors disrupts the population profile of intestinal microbiota. To date, such studies have primarily focused on prolonged stressors (repeated across several days) and have assessed fecal bacterial populations. It is not known whether shorter stressors can also impact the microbiota, and whether colonic mucosa-associated populations can also be affected. The mucosa-associated microbiota exist in close proximity to elements of the host immune system and the two are tightly interrelated. Therefore, alterations in these populations should be emphasized. Additionally, stressors can induce differential responses in anxiety-like behavior and corticosterone outputs in variant strains of mice. Thus, whether stressor exposure can have contrasting effects on the colonic microbiota in inbred C57BL/6 mice and outbred CD-1 mice was also examined.ResultsIn the present study, we used high throughput pyrosequencing to assess the effects of a single 2-hour exposure to a social stressor, called social disruption (SDR), on colonic mucosa-associated microbial profiles of C57BL/6 mice. The data indicate that exposure to the stressor significantly changed the community profile and significantly reduced the relative proportions of two genera and one family of highly abundant intestinal bacteria, including the genus Lactobacillus. This finding was confirmed using a quantitative real-time polymerase chain reaction (qPCR) technique. The use of qPCR also identified mouse strain-specific differences in bacterial abundances. L. reuteri, an immunomodulatory species, was decreased in stressor-exposed CD-1 mice, but not C57BL/6 mice.ConclusionsThese data illustrate that stressor exposure can affect microbial populations, including the lactobacilli, that are closely associated with the colonic mucosa. Because the lactobacilli can have beneficial effects on human health, stressor-induced reductions of their population could have important health implications.

Early experience of a novel-environment in isolation primes a fearful phenotype characterized by persistent amygdala activation

Prolonged maternal separation (MS) activates the neonate's hypothalamus-pituitary-adrenal axis causing elevated basal and stress-induced corticosterone levels that may initiate amygdala-dependent fear learning. Here we test the hypothesis that the adult fearful phenotype is programmed by the pup's stressful experience during prolonged MS rather than by prolonged maternal absence per se. For this purpose, Wistar rat pups were exposed, on postnatal-day (pnd) 3, to: (i) repeated-MS in home-environment (HOME-SEP), 8h-MS daily for three days with the pups remaining together in the home-cage; (ii) repeated-MS in a novel-environment (NOVEL-SEP), with the same separation procedure, but now the pups were individually housed in a novel-environment during the 8h dam's absence; (iii) repeated handling, which consisted of daily brief (15 min instead of 8h) MS in the home-altogether or in a novel-environment individually (HOME-HAN and NOVEL-HAN, respectively); (iv) no-separation/no-handling (NON-SEP/NON-HAN) control condition, in which pups were left undisturbed in their home-cage. Compared to HOME-SEP rats, the NOVEL-SEP rats showed one day after the last MS enhanced stress-induced amygdala c-Fos expression and ACTH-release, despite of reduced adrenal corticosterone secretion. The higher amygdala c-Fos expression, ACTH-release and reduced corticosterone output observed postnatally, persisted into adulthood of the NOVEL-SEP animals. Behaviorally, NOVEL-SEP juvenile rats displayed deficits in social play, had intact spatial memory in the peri-pubertal period and showed more contextual fear memory compared to HOME-SEP in adulthood. Finally, NOVEL-HAN, compared to HOME-HAN, displayed increased stress-induced corticosterone output, no deficits in social play and reduced contextual fear. In conclusion, programming of an adult fearful phenotype linked to amygdala priming develops if pups are repeatedly isolated from peers in a novel-environment, while away from the dam for a prolonged period of time.

Effect of reproductive status on hypothalamic–pituitary–adrenal (HPA) activity and reactivity in male California mice (Peromyscus californicus)

Previous studies indicate that reproductive condition can alter stress response and glucocorticoid release. Although the functional significance of hypothalamic–pituitary–adrenal (HPA) axis modulation by breeding condition is not fully understood, one possible explanation is the behavior hypothesis, which states that an animal's need to express parental behavior may be driving modulation of the HPA axis. This possibility is consistent with findings of blunted activity and reactivity of the HPA axis in lactating female mammals; however, effects of reproductive status on HPA function have not been well characterized in male mammals that express parental behavior. Therefore, we tested this hypothesis in the monogamous and biparental California mouse. Several aspects of HPA activity were compared in males from three reproductive conditions: virgin males (housed with another male), non-breeding males (housed with a tubally ligated female), and first-time fathers (housed with a female and their first litter of pups). In light of the behavior hypothesis we predicted that new fathers would differ from virgin and non-breeding males in several aspects of HPA function and corticosterone (CORT) output: decreased amplitude of the diurnal rhythm in CORT, a blunted CORT increase following predator-odor stress, increased sensitivity to glucocorticoid negative feedback, and/or a blunted CORT response to pharmacological stimulation. In addition, we predicted that first-time fathers would be more resistant to CORT-induced suppression of testosterone secretion, as testosterone is important for paternal behavior in this species. We found that virgin males, non-breeding males and first-time fathers did not display any CORT differences in diurnal rhythm, response to a predator-odor stressor, or response to pharmacological suppression or stimulation. Additionally, there were no differences in circulating testosterone concentrations. Adrenal mass was, however, significantly lower in new fathers than in virgin or non-breeding males. These results suggest that the behavior hypothesis does not explain HPA function across reproductive conditions in male California mice.

Short-term voluntary exercise in the rat causes bone modeling without initiating a physiological stress response

Recent research has revealed a neuroendocrine connection between the skeleton and metabolism. Exercise alters both bone modeling and energy balance and may be useful in further developing our understanding of this complex interplay. However, research in this field requires an animal model of exercise that does not cause a physiological stress response in the exercised subjects. In this study, we develop a model of short-term voluntary exercise in the female rat that causes bone modeling without causing stress. Rats were randomly assigned to one of three age-matched groups: control, tower climbing, and squat exercise (rising to an erect bipedal stance). Exercise for 21 days resulted in bone modeling as assessed by peripheral quantitative computed tomography. Fecal corticosterone output was used to assess physiological stress at three time points during the study (preexercise, early exercise, and late in the exercise period). There were no differences in fecal corticosterone levels between groups or time points. This model of voluntary exercise in the rat will be useful for future studies of the influence of exercise on the relationship between skeletal and metabolic health and may be appropriate for investigation of the developmental origins of those effects.

Adiponectin and adiponectin receptor system in the rat adrenal gland: Ontogenetic and physiologic regulation, and its involvement in regulating adrenocortical growth and steroidogenesis

Adiponectin (ADN) is a regulatory peptide secreted mostly by adipose tissue and acting via two receptors: AdipoR1 and AdipoR2. Our aim was to investigate expression of adiponectin system genes in the rat adrenal gland as well as its ontogenetic and physiological control. Furthermore, we examined the effects of acute and prolonged activation of HPA axis on ADN system in adipose tissue. By means of QPCR, ADN and AdipoR1 expression was demonstrated in rat adrenal cortex both at mRNA and protein levels, while AdipoR2 could only be detected at mRNA levels. ADN expression level was significantly upregulated in a developing and regenerating adrenal cortex. Globular domain of adiponectin at 10 −9 M stimulated corticosterone output and BrdU incorporation by cultured rat adrenocortical cells. Moreover, both acute (ACTH and ether stress) and prolonged (ACTH) adrenal stimulation resulted in lowered ADN levels, while expression of AdipoR1 and AdipoR2 was upregulated by the acute treatment. Depending on its site of origin, visceral (VAT) or subcutaneous (SAT) adipose tissue responded differently to alterations in HPA axis. VAT expression of ADN and its receptors remained almost unchanged by experimental manipulations. In SAT, on the other hand, expression of ADN and AdipoR2 was markedly increased by ACTH treatment and stress, while dexamethasone suppressed ADN and AdipoR1 mRNA levels. The results of this study provide new evidence for direct and indirect interactions between adipokines and HPA axis.

Frustration and perseveration in stereotypic captive animals: Is a taste of enrichment worse than none at all?

Stereotypic behaviours are common in animals in impoverished housing, arising from two complementary processes: (1) thwarted attempts to perform motivated behaviours; (2) forebrain dysfunction impeding normal behavioural inhibition. When enriched animals are moved to impoverished housing, they are sometimes protected against developing stereotypic behaviour, but in other cases become even more stereotypic than animals housed lifelong without enrichment. Negative contrast-induced frustration must occur in both scenarios. We hypothesise that sustained behavioural responses to this frustration are prevented in the former by normalised forebrain function, but exacerbated in the latter by forebrain dysfunction. ICRCD-1 mice reared in enriched or standard cages were re-caged at 3 months to standard conditions. Here, previously-enriched mice became far more stereotypic than mice reared from birth in such conditions. To investigate the role of frustration, we assessed both corticosterone output and motivation (break-point) to regain enrichments. We also assessed perseveration via extinction learning. As predicted, previously-enriched mice were as perseverative as standard-raised mice, and frustration seemed to play a causal role in their exacerbated stereotypic behaviour. Previously-enriched mice showed higher motivations to access enrichments, and only in this group did these correlate with corticosterone levels after re-caging; furthermore only in previously-enriched mice did corticosterone responses to re-caging predict stereotypic behaviour 30 days later (males only). All results need replicating and further investigation. However, they suggest for the first time that individual risk factors related to the HPA axis predict stereotypic behaviour following enrichment-removal, and that previously-enriched mice have lasting motivational differences from standard-raised mice, suggesting sustained behavioural effects related to the frustration of enrichment-loss.

The effect of CAG repeat polymorphism in the glucocorticoid receptor on stress responses of mice exposed to water-immersion restraint stress

Exposure to stress activates the hypothalamus-pituitary-adrenal (HPA) axis, which is followed by an increase in production of its end product, corticosterone, considered to be the most important glucocorticoid (GC) in rodents. Glucocorticoid receptor (GR) signaling has been suggested as a potential mechanism responsible for the pathogenesis of many clinical disorders. Here, we investigated the involvement of the GR polymorphism in stress response. ICR mice were screened by genomic PCR, bred, and divided into 3 groups according to the GR polymorphism, GRwt/wt, GRwt/Qn, and GRQn/Qn. Mice were exposed to water-immersion restraint stress (WRS), and then examined for gastric mucosal lesions, serum corticosterone, serum cytokines and serum Hsp70 levels. Male mice with GRQn/Qn exhibited a significantly greater gastric lesion index than those with GRwt/wt at 6 h of WRS. Stress-induced corticosterone output achieved peak levels at 3 h, after which it was downregulated. The serum level in the control group was GRwt/wt >GRwt/Qn >GRQn/Qn, whereas the order at 6 h of WRS was reversed, GRQn/Qn >GRwt/Qn >GRwt/wt, suggesting that the GRwt allele responded rapidly to stress. The IL-6 levels of each polymorphic line increased at 3 h and particularly at 6 h. On the contrary, the IL-10 levels in GRwt/wt and GRwt/Qn increased following exposure to WRS, whereas that in GRQn/Qn showed no change. The Hsp70 levels in mice with GRQn allele particularly increased at 6 h of WRS, and the concentration in GRQn/Qn significantly increased as compared to that in GRwt/wt. These results suggest that the GR gene polymorphism has a significant impact on the stress-induced output, including the gastric lesion index, corticosterone, cytokines, and Hsp70 levels in serum. The present study provides insights into the role of GR in individual responses to stress.

Testosterone and melatonin effects on adrenal cortex of orchiectomized and pinealectomized rats.

The study aimed to examine the effects of pinealectomy (PX) and melatonin on adrenal cortex of orchiectomized rats and to explain whether the testosterone action on adrenal cortex is modified by the pineal gland. Neither PX (53 days) nor melatonin administration (10 days) had an effect on the structure as well as on the studied functional parameters of the adrenal gland--corticosterone output by homogenates, 11 beta-hydroxylase and 5 alpha-reductase activities, liver corticosterone metabolism and serum corticosterone level. On the contrary, PX of 14 months duration resulted in an increase in 5 alpha-reductase activity. Testosterone administration into orchiectomized rats lowered corticosterone output and liver corticosterone metabolism and these effects were unaffected by PX or melatonin. Testosterone lowered 5 alpha-reductase and 11 beta-hydroxylase activities, the effects not modified by PX, however, after melatonin administration activities of these enzymes were similar as in orchiectomized rats. Obtained results cast doubts on a physiological role of the pineal gland and its principal hormone--melatonin--in regulation of adrenocortical structure and secretory activity and suggest that testosterone action on the rat adrenal cortex is not modified by the pineal gland.

Expression of prepro-ghrelin and related receptor genes in the rat adrenal gland and evidences that ghrelin exerts a potent stimulating effect on corticosterone secretion by cultured rat adrenocortical cells

The orexigenic peptide ghrelin (GHREL) and obestatin (OBS) originate from the same peptide precursor, preproghrelin (ppGHREL). Apart from orexigenic effect, GHREL also regulates neuroendocrine function. We investigated GHREL and OBS effects on corticosterone secretion by freshly isolated and cultured rat adrenocortical cells. Classic RT-PCR revealed the presence of ppGHREL, GHS-R1a, GPR39v1 and GPR39v2 and GOAT4 (ghrelin O-acyl transferase) mRNAs in rat adrenals and cultured for 4 days rat adrenocortical cells. Expression of ppGHREL, GHS-R1a, and GOAT genes was notably higher in the cortex than in medulla. High expression level of GOAT gene was found in the zona glomerulosa, while expression level of both GPR39v1 and GPR39v2 genes was similar in adrenal cortical zones and in medulla. In freshly isolated cells neither GHREL nor OBS had an effect on corticosteroid output. Prolonged exposure of cultured cells to GHREL resulted in a potent, comparable to ACTH, stimulating effect of GHREL on corticosterone secretion. Prolonged exposure to OBS was ineffective. Neither GHREL nor OBS had any effect on proliferation of studied cells, while ACTH notably lowered it. GHREL down regulated GHS-R1a gene expression while both ACTH and GHREL stimulated expression level of GPR39v1 gene. Expression of CYP11A1 gene was notably stimulated and that of StAR gene remained unaffected by ACTH or GHREL. Thus, our study is the first to demonstrate direct stimulating effect of GHREL on corticosterone output by cultured rat adrenocortical cells. This stimulating action differs from that evoked by ACTH and is not dependent on the presence of functional ACTH receptor.

Cerebellin and des-cerebellin exert ACTH-like effects on corticosterone secretion and the intracellular signaling pathway gene expression in cultured rat adrenocortical cells - DNA microarray and QPCR studies

Precerebellins (Cbln) belong to the C1q/TNF superfamily of secreted proteins which have diverse functions. They are abundantly expressed in the cerebellum, however, three of them are also expressed in the rat adrenal gland. All members of the Cbln family form homomeric and heteromeric complexes with each other in vitro and it was suggested that such complexes play a crucial role in normal development of the cerebellum. The aim of our study was to investigate whether Cbln1-derived peptides, cerebellin (CER) and des-Ser1-cerebellin (desCER) are involved in regulating biological functions of rat adrenocortical cells. In the primary culture of rat adrenocortical cells, 24 h exposure to CER or desCER notably stimulated corticosterone output and inhibited proliferative activity and similar effects were evoked by ACTH. To study gene transcript regulation by CER, desCER and ACTH, we applied Oligo GEArray DNA Microarray: Rat Signal Transduction Pathway Finder. In relation to the control culture, 13 of the 113 transcripts present on the array were differentially expressed. These transcripts were either up- or down-regulated by ACTH and/or CER or desCER treatment. Validation of DNA Microarray data by QPCR revealed that only 5 of 13 genes studied were differentially expressed. Of those genes, Fos and Icam1 were up-regulated and Egr1 was down-regulated by ACTH, CER and desCER. The remaining two genes, Fasn (insulin signaling pathway) and Hspb1 (HSP27) (stress signaling pathway), were regulated only by CER and desCER, but not by ACTH. Thus, both CER and desCER have effects similar to and different from corticotrophin on the intracellular signaling pathway gene expression in cultured rat adrenocortical cells.

The effects of ACTH, phytoestrogens and estrogens on corticosterone secretion by gander adrenocortical cells in breeding and nonbreeding seasons

The aim of this study was to investigate the effects of ACTH, phytoestrogens (genistein, daidzein, biochanin A and coumestrol), and animal estrogens (estradiol and estrone) on corticosterone secretion by isolated adrenocortical cells of the ganders in breeding (April) and nonbreeding seasons (July). ACTH stimulated corticosterone output in the breeding season. In July (photorefractoriness and postbreeding molt) ACTH had no effect on corticosterone production. Coumestrol reduced corticosterone secretion by the cells obtained in nonbreeding season. Other examined phytoestrogens did not affect corticosterone production. Estrogens showed differentiated effects. Estradiol stimulated the corticosterone output in breeding season; estrone inhibited corticosterone release in July. The season can probably affect sensitivity of isolated gander adrenal cells, especially to ACTH. It seems that goose adrenocortical cells, in contrast to the mammalian cells, can be weakly sensitive to phytoestrogens.

Photoperiodically-induced changes in hypothalamic–pituitary–adrenal axis sensitivity in captive house sparrows (Passer domesticus)

We used captive house sparrows (Passer domesticus) to identify regulatory mechanisms underlying seasonal (mimicked by changes in photoperiod) and diel differences in corticosterone output. Corticosterone responses were measured during three simulated seasons: short-day and long-day photoperiods and while birds underwent a pre-basic molt. Under all three conditions we tested for adrenal sensitivity by injecting exogenous ACTH, for pituitary sensitivity by injecting corticotropin-releasing factor (CRF) and arginine vasotocin (AVT), and for diel changes by repeating the injections during the day and at night. The daytime adrenal sensitivities were greatest on long days, lower on short days, and lowest during molt. These data suggest that reductions in either adrenal sensitivity to ACTH and/or capacity to secrete corticosterone could explain lowered endogenous corticosterone titers during molt. Furthermore, adrenal sensitivity to ACTH and pituitary sensitivity to AVT appeared to be greatest at night. This suggests that both the adrenal's sensitivity to the ACTH signal and the pituitary's capacity to secrete ACTH might provide a mechanism allowing for diel changes in corticosterone titers. This differs substantially from what is known about diel regulation in rodents. Taken together, these data provide further evidence that there are complex regulatory mechanisms controlling diel and seasonal changes in corticosterone titers in birds.