Corticosterone Production Research Articles

Corticosteroid therapy for patients in septic shock: Some progress in a difficult decision

Objectives: Reversible adrenal insufficiency has been frequently diagnosed in critically ill patients with sepsis who have either low basal cortisol levels or low cortisol responses to adrenocorticotrophic hormone (ACTH) stimulation. It is generally accepted that a phenomenon called contributes to immunosuppression during sepsis. The present study was to investigate whether endotoxin tolerance occurs in the adrenal gland, leading to hyporesponsiveness of adrenal gland during sepsis. Design: Controlled laboratory experiment. Setting: University research laboratory. Subjects: Sprague-Dawley male rats 200-250 g, and primary isolated adrenal fasciculata-reticularis cells. Interventions: Rats received intra-arterial injection of purified lipopolysaccharide (LPS, 0.5 mg/kg) through indwelling femoral arterial catheters, and 24 h later the adrenocortical sensitivity to exogenous ACTH (10 ng/kg) was detected. Primary F/R cells were pretreated with LPS at 0.1-100 ng/mL or with ACTH at 0.01―10 ng/mL, and then challenged, in fresh media, with 1 μg/mL LPS or 10 ng/mL ACTH. Measurements and Main Results: Toll-like receptor 4 were expressed in adrenal gland and primary fasciculata-reticularis cells. Plasma corticosterone response to ACTH was decreased in rats receiving preinjection of LPS. LPS pretreatment caused a significant decrease in corticosterone production in response to subsequent ACTH and LPS stimulation in primary fasciculata-reticularis cells. LPS pretreatment inhibited ACTH- and LPS-induced expression of steroid metabolizing enzymes. LPS significantly decreased toll-like receptor 4 and ACTH receptor expression. Conclusions: Pre-exposure to LPS resulted in hyporesponsiveness to ACTH stimulation in rats. In vitro, LPS pretreatment impaired corticosterone production of F/R cells in response to ACTH and LPS, which was associated with decreased expression of synthetic enzymes required for corticosterone production. Our results indicate that endotoxin tolerance of adrenal gland is one of mechanisms for adrenocortical insufficiency during sepsis.

Genetic modifications of mouse proopiomelanocortin peptide processing

Pro-opiomelanocortin (POMC) is a prohormone which undergoes extensive tissue and cell specific post-translational processing producing a number of active peptides with diverse biological roles ranging from control of adrenal function to pigmentation to the regulation of feeding. One approach to unraveling the complexities of the POMC system is to engineer mouse mutants which lack specific POMC peptides. We describe here the design, generation, validation, and preliminary analysis of one such partial POMC mutant specifically lacking α-MSH. In contrast to POMC null mutant mice, mice lacking α-MSH in the presence of all other POMC peptides maintain adrenal structures and produce corticosterone comparable to wildtype littermates; however, they still have decreased levels of aldosterone, as found in POMC null mutant mice. Our findings demonstrate that α-MSH is not needed for maintenance of adrenal structure or for corticosterone production, but is needed for aldosterone production. These data demonstrate that mouse strains generated with precise genetic modifications of POMC peptide processing can answer questions about POMC peptide function. Further analysis of this and additional strains of mice with modified POMC peptide processing patterns will open up a novel avenue for studying the roles of individual POMC peptides.

Chronic Effects of Propylthiouracil on Metabolism and Secretion of Adrenocorticotropin and Corticosterone in Male Rats

Background: Propylthiouracil (PTU) is a thioamide drug for treating hyperthyroidism. We have already found that chronic administration of PTU decreases the level of plasma corticosterone and attenuates both the activities of 11β- hydroxylase and the generation of cyclic adenosine monophosphate (cyclic AMP) in rat zona fasciculata-reticularis (ZFR) cells. The present study aimed to investigate the chronic effects of PTU on metabolism, adrenocorticotropic hormone (ACTH) and corticosterone secretion. Methods: Male rats were randomly divided into four groups and injected subcutaneously with saline, PTU, thyroxine (T4) or PTU plus T4 once daily for 2 weeks. For metabolism studies, the body weight, food intake, water intake, urine volume, and feces weight of every rat were recorded daily. The rats were sacrificed after treatment, and the plasma thyroid stimulating hormone (TSH), ACTH, and corticosterone were measured. ZFR cells were separated from adrenal glands and then stimulated by forskolin (an activator of adenylyl cyclase, 10^(-5) M), 8-bromo-cAMP (8-Br-cAMP, a permeable analogue of cAMP, 10^(-5) M), ACTH (a stimulator to increase corticosterone secretion, 10^(-9) M), and deoxycorticosterone (DOC, a steroidogenic precursor of corticosterone, 10^(-5) M) in order to investigate the alternation of corticosterone secretion and enzyme activity. Results: Chronic administration of PTU decreased food intake and feces weight but increased urine volume. The concentration of plasma TSH increased after chronic PTU treatment. PTU also suppressed release of ACTH and decreased production of corticosterone. After the isolation of ZFR cells from PTU and PTU plus T4-treated rats, both basal and evoked levels of corticosterone release were enhanced. Conclusion: Chronic administration of PTU inhibited pituitary gland from releasing ACTH, and this reaction led to decrease in corticosterone production in vivo. PTU treatment might upregulate downstream messengers of ACTH signal pathway in ZFR cells, thus enhancing release of corticosterone in vitro.

Effect of diabetes on enzymes involved in rat hepatic corticosterone production

Numerous studies have explored the etiologic or permissive role of 11β-hydroxysteroid dehydrogenase (11β-HSD1) in obesity and Type 2 diabetes, biochemical conditions often with concurrent hyperinsulinism. In contrast, there are limited data on the effect of insulin deficiency (i.e. Type 1 diabetes) on 11β-HSD1 or endoplasmic reticulum enzymes that generate the reduced pyridine cofactor NADPH. Thus, the aim of the present study was to examine the effect of insulin-deficient, streptozotozin diabetes on key microsomal enzymes involved in rat hepatic corticosterone production. After rats had been rendered diabetic with streptozotocin and some had been treated with insulin (2-6 units, s.c., long-acting insulin once daily) for 7 days, hepatic microsomes were isolated. Serum corticosterone and fructosamine were obtained premortem. Intact microsomes were incubated in vitro and 11β-HSD1, hexose-6-phosphate dehydrogenase (H6PDH), and isocitrate dehydrogenase (IDH) measured. Although diabetes markedly altered body weight gain and serum protein glycosylation (assessed by fructosamine), there was no significant change in hepatic 11β-HSD1 reductase activity, with or without insulin treatment. However, serum corticosterone levels were significantly correlated with 11β-HSD1 reductase activity when all groups were analyzed together (P < 0.05). Untreated diabetes modified (P < 0.01) two hepatic microsomal NADPH-generating enzymes, namely H6PDH and IDH, resulting in a 37% decrease and 14% increase in enzyme levels, respectively. Consistent with most in vivo studies, chronic insulin deficiency with attendant hyperglycemia does not significantly modify hepatic 11β-HSD1 reductase activity, but does alter the activity of two microsomal enzymes coupled with pyridine cofactors.

Production of Aldosterone and Corticosterone in Polycystic Ovary Syndrome (PCOS) Rats.

Production of Aldosterone and Corticosterone in Polycystic Ovary Syndrome (PCOS) Rats.

The β-Human Chorionic Gonadotropin-Related Peptide LQGV Exerts Anti-Inflammatory Effects through Activation of the Adrenal Gland and Glucocorticoid Receptor in C57BL/6 Mice

The systemic inflammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin β-chain-related tetrapeptide leucine-glutamine-glycine-valine (LQGV) reduces hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully understood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-α and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that LQGV stimulates corticosterone production. Moreover, by using specific pharmacological inhibitors of the adrenocorticotropic hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the β-human chorionic gonadotropin-related tetrapeptide LQGV stimulates adrenal glucocorticosteroid production through activation of the ACTH receptor with consequent glucocorticoid receptor activation and immunosuppression in C57BL/6 mice.

High levels of mineralocorticoids in preovulatory follicular fluid could contribute to oocyte development

To identify aldosterone and precursors in ovarian follicles and to relate levels of mineralocorticoids to previously described renin-angiotensin system follicular fluid content. Experimental. Academic laboratory and affiliated large private practice. Women undergoing oocyte retrieval for in vitro fertilization (IVF). The concentrations of mineralocorticoids were measured in plasma and follicular fluid. Granulosa cell mRNA expression and oocyte receptor content were evaluated. High concentrations of preovulatory aldosterone and corticosterone were measured in follicular fluid (419.5±122.2 and 218,383±124,143 pg/mL, respectively). Increased mineralocorticoid levels are found in follicular fluid compared with in plasma and in large follicles compared with in small. Plasma aldosterone levels increase before ovulation. Granulosa cell gene expression that promotes aldosterone production and accumulation of corticosterone is increased in younger patients. Aldosterone receptors are localized to the surface of human oocytes. High levels of aldosterone and its precursor, corticosterone, were found in ovarian follicles. This combined with the presence of aldosterone receptors on oocytes suggests a possible role for aldosterone in oocyte development.

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A(2) (GX KO). These mice have approximately 80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A(2) (sPLA(2)), but not a catalytically inactive mutant form of GX sPLA(2), significantly reduced steroid production 30-40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA(2) inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA(2)-overexpressing Y1 cells, ruling out a role for this sPLA(2) receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was approximately 2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA(2). Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA(2) antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA(2) is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.

1α,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R cells

The current study presents data indicating that 1α,25-dihydroxyvitamin D3 affects the production of hormones and expression of crucial steroidogenic enzymes in the human adrenocortical cell line NCI-H295R. This cell line is widely used as a model for adrenal steroidogenesis. Treatment of the cells with 1α,25-dihydroxyvitamin D3 suppressed the levels of corticosterone, aldosterone, DHEA, DHEA-sulfate and androstenedione in the culture medium. In order to study the mechanisms behind this suppression of hormone production, we investigated the effects of 1α,25-dihydroxyvitamin D3 on important genes and enzymes controlling the biosynthesis of adrenal hormones. The mRNA levels were decreased for CYP21A2 while they were increased for CYP11A1 and CYP17A1. No significant changes were observed in mRNA for CYP11B1, CYP11B2 or 3β-hydroxysteroid dehydrogenase (3βHSD). In similarity with the effects on mRNA levels, also the endogenous enzyme activity of CYP21A2 decreased after treatment with 1α,25-dihydroxyvitamin D3. Interestingly, the two CYP17A1-mediated activities were influenced reciprocally — the 17α-hydroxylase activity increased whereas the 17,20-lyase activity decreased. The current data indicate that the 1α,25-dihydroxyvitamin D3-mediated decrease in corticosterone and androgen production is due to suppression of the 21-hydroxylase activity by CYP21A2 and the 17,20-lyase activity by CYP17A1, respectively. In conclusion, the current study reports novel findings on 1α,25-dihydroxyvitamin D3-mediated effects on hormone production and regulation of genes and enzymes involved in steroidogenesis in the adrenocortical NCI-H295R cell line, a model for human adrenal cortex.

NF-κB activity affects learning in aversive tasks: Possible actions via modulation of the stress axis

The role of altered activity of nuclear factor κB (NF-κB) in specific aspects of motivated behavior and learning and memory was examined in mice lacking the p50 subunit of the NF-κB/rel transcription factor family. Nfkb1-deficient mice are unable to produce p50 and show specific susceptibilities to infections and inflammatory challenges, but the behavioral phenotype of such mice has been largely unexamined, owing in large part to the lack of understanding of the role of NF-κB in nervous system function. Here we show that Nfkb1 (p50) knockout mice more rapidly learned to find the hidden platform in the Morris water maze than did wildtype mice. The rise in plasma corticosterone levels after the maze test was greater in p50 knockout than in wildtype mice. In the less stressful Barnes maze, which tests similar kinds of spatial learning, the p50 knockout mice performed similarly to control mice. Adrenalectomy with corticosterone replacement eliminated the differences between p50 knockout and wildtype mice in the water maze. Knockout mice showed increased levels of basal anxiety in the open-field and light/dark box tests, suggesting that their enhanced escape latency in the water maze was due to activation of the stress (hypothalamic–pituitary–adrenal) axis leading to elevated corticosterone production by strongly but not mildly anxiogenic stimuli. The results suggest that, as in the immune system, p50 in the nervous system normally serves to dampen NF-κB-mediated intracellular activities, which are manifested physiologically through elevated stress responses to aversive stimuli and behaviorally in the facilitated escape performance in learning tasks.

Abstract DD01-03: TAK-700, an inhibitor of 17,20-lyase

Abstract Background: Recent studies suggest that residual adrenal androgen after castration could be responsible for the progression of castration-resistant prostate cancer (CRPC). To develop an effective therapy for CRPC that inhibits the production of adrenal and testicular androgens, we searched for a novel, non-steroidal, selective, and potent inhibitor of 17,20-lyase, a key enzyme in androgen synthesis, and identified TAK-700 as a clinical candidate. Methods: Enzyme assay was conducted using radiolabeled substrates. The enzyme reaction products were separated by thin layer chromatography and the radioactivity was measured by a bio-image analyzer. We assessed the activity and specificity of TAK-700 on androgen production in vitro in monkey adrenal cells and human adrenocortical tumor cell line, using a radioimmunoassay. Using a single dose of [14C]TAK-700 1 mg/kg, we investigated the pharmacokinetics and bioavailability of TAK-700 in cynomolgus monkeys, and assessed the effects of TAK-700 7.5 and 15 mg/kg twice daily (BID) for 7 days on serum androgen levels in castrated and intact cynomolgus monkeys. Results: TAK-700 potently inhibited the activities of human and monkey 17, 20-lyase (CYP17A1) with IC50 values of 140 and 27nmol/L, respectively. The inhibitory activity of TAK-700 on 17-hydroxylase was lower than that on 17, 20-lyase in all the enzyme preparations. IC50 values of ketoconazole and abiraterone for human 17, 20-lyase were 110 and 27nmol/L, respectively. TAK-700 did not inhibit monkey 11-hydroxylase up to 10μmol/L. In addition, IC50 values of TAK-700 for all CYP-specific activities (drug-metabolizing enzymes) examined were higher than 10 μmol/L. TAK-700 suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 110, 130, 310, &amp;gt;100,000 and 4,400nmol/L, respectively. The IC50 value for cortisol production was only 3-fold higher than that for DHEA production in monkeys, reflecting low specificity of inhibition between monkey 17, 20-lyase and 17-hydroxylase activity (IC50 values: 27 vs 38nmol/L). Ketoconazole suppressed the production of DHEA, androstenedione, cortisol, corticosterone and aldosterone by monkey adrenal cells with IC50 values of 340, 580, 340, 1,500 and 350nmol/L, respectively. TAK-700 inhibited the production of dehydroepiandrosterone (DHEA) and cortisol in human adrenocortical tumor line NCI-H295R cells with IC50 values of 37 and 990nmol/L, respectively, showing about 27-fold selectivity for DHEA production. Ketoconazole inhibited the production of DHEA and cortisol with IC50 values of 330 and 480nmol/L (selectivity 1.5-fold), respectively, and abiraterone inhibited the production of DHEA and cortisol with IC50 values of 2.7 and 23nmol/L (selectivity: 8.5-fold), respectively. TAK-700 showed the highest specificity of inhibition for DHEA production among these 3 compounds. After oral administration of [14C]TAK-700 1 mg/kg, maximum plasma concentration (Cmax) was 0.147 μg/mL, time to Cmax (Tmax) was 1.7 hours, elimination half-life (t1/2) was 3.8 hours, and exposure (area under the plasma concentration-time curve in 24 hours, AUC0-24h) was 0.727 μg·h/mL. Oral bioavailability of TAK-700 in monkeys was 70.5%. Oral dosing of TAK-700 7.5 and 15 mg/kg BID rapidly (within 1 day) suppressed serum levels of the androgens dehydroepiandrosterone (DHEA) and testosterone and of cortisol in both castrated and gonadally intact monkeys. While suppression of DHEA and cortisol levels persisted during the 7-day treatment period in castrated monkeys, suppression of testosterone was attenuated from the second day of treatment, possibly due to activation of the hypothalamus-pituitary-gonad axis. TAK-700 had a milder suppressive effect on cortisol levels than on DHEA levels. During the treatment period, mean DHEA levels decreased to 9.0% and 6.9% of baseline levels in castrated monkeys treated with TAK-700 7.5 and 15 mg/kg, respectively. The mean cortisol levels decreased to 26.0% and 17.3% of baseline levels with TAK-700 7.5 and 15 mg/kg, respectively. In vitro results have shown that the specificity of TAK-700 suppression of DHEA production over cortisol production is 10-fold greater in human adrenal-origin cells than in monkey adrenal cells. Therefore, the impact of TAK-700 on human serum cortisol levels may be smaller than that in monkeys. Conclusion: As TAK-700 potently and selectively inhibited 17, 20-lyase activity and DHEA production by H295R cells and suppressed serum androgen levels in castrated monkeys, TAK-700 may be effective for at least some types of CRPC, and clinical evaluation of TAK-700 is now underway in patients with CRPC. Citation Format: Masuo Yamaoka, Takahito Hara, Hiroshi Miki, Takenori Hitaka, Tomohiro Kaku, Naohiro Kawaguchi, Hitomi Yamasaki, Toshiyuki Takeuchi, Akihiro Tasaka, Masami Kusaka. TAK-700, an inhibitor of 17,20-lyase [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr DD01-03

Androgenic Influence on Serotonergic Activation of the HPA Stress Axis

The higher incidence of stress-mediated affective disorders in women may be a function of gonadal hormone influence on complex interactions between serotonin and neural circuits that mediate the hypothalamic-pituitary-adrenal (HPA) stress axis. The paraventricular nucleus of the hypothalamus (PVN) receives serotonergic innervation, and selective serotonin reuptake inhibitors such as citalopram activate the HPA axis independent of stress. We have previously demonstrated that the magnitude of this serotonergic activation was greater in females and was attenuated by testosterone administration; however, the potential central sites of action where androgens reduce these serotonergic effects have not been determined. Therefore, we examined a time course of corticosterone production and used central c-Fos protein levels to assay neuronal activation in stress-related brain regions in female, male, and gonadectomized male mice after an acute citalopram injection (15 mg/kg). In the hippocampus, c-Fos-immunoreactivity was greater in males than in females or gonadectomized males. This same pattern emerged in the lateral septum after vehicle and gonadectomy reversed the effect of citalopram. These regions are important for inhibitory influences on the PVN, and accordingly, hippocampal c-Fos levels were negatively correlated with corticosterone production. No sex differences in c-Fos were detected in the PVN, cingulate cortex, or paraventricular thalamus in response to vehicle or citalopram. These data support brain region-specific regulation of the HPA axis where sex differences may be mediated partly through androgen enhancement of signaling in inhibitory regions.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor α and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Sex Differences in the Serotonergic Influence on the Hypothalamic-Pituitary-Adrenal Stress Axis

Appropriate interactions between serotonin (5-HT) and stress pathways are critical for maintaining homeostasis. Dysregulation of hypothalamic-pituitary-adrenal (HPA) stress axis is a common feature in affective disorders in which an involvement of 5-HT neurocircuitry has been implicated in disease vulnerability and treatment responsiveness. Because there is a greater prevalence of affective disorders in women, sex differences in the 5-HTergic influence on stress pathways may contribute to disease disparity. Therefore, our studies compared stress or citalopram-induced corticosterone levels in male and female mice. To determine whether sex-dependent HPA axis responsiveness was mediated by the difference in testosterone levels, testosterone-treated females were also examined. Gene expression patterns in 5-HTergic and stress neurocircuitry were analyzed to determine sites of potential sex differences and mechanisms of testosterone action. As expected, restraint stress corticosterone levels were higher in intact females and were masculinized by testosterone. Interestingly, citalopram administration independent of stress resulted in a greater corticosterone response in females, which was also masculinized by testosterone. Analyses along the 5-HT-HPA axis revealed sex differences including greater pituitary 5-HT receptors and adrenal weights in females. Moreover, in stress-regulatory regions, we found sex differences in glucocorticoid receptor and glutamic acid decarboxylase expression supportive of greater inhibitory modulation and feedback potential in males. Taken together, these data suggest that multiple sites related to 5-HTergic stimulation, corticosterone production, and negative feedback of HPA neurocircuitry combine to produce higher female stress responsiveness. These studies support a potential for sex-specific involvement of 5-HT and stress pathways in the etiology of affective disorders.

GLUCOCORTICOID TREATMENT-EFFECT ON ADRENAL MEDULLARY CATECHOLAMINE PRODUCTION

Glucocorticoid and epinephrine are important stress hormones secreted from the adrenal gland during critical illness. Adrenal glucocorticoid stimulates phenylethanolamine N-methyltransferase (PNMT) to convert norepinephrine to epinephrine in the adrenal medulla. Glucocorticoid is sometimes used in catecholamine-resistant septic shock in critically ill patients. By suppressing adrenal glucocorticoid production, glucocorticoid therapy might also reduce the secretion of epinephrine during stress. To investigate this, we used a mouse model subjected to glucocorticoid therapy under basal conditions (experiment 1) and during stress (experiment 2). In experiment 1, pellets containing 0% to 8% dexamethasone were implanted subcutaneously in mice for 4 weeks. In experiment 2, animals received 14 days of intraperitoneal injections of normal saline, low- or high-dose dexamethasone, followed by 2 h of restraint. We found that in experiment 1, adrenal corticosterone did not differ with dexamethasone treatment. Phenylethanolamine N-methyltransferase messenger RNA levels and adrenal catecholamines were highest in the 8% dexamethasone group. Compared with experiment 1, restrained control mice in experiment 2 had high adrenal corticosterone, which decreased with dexamethasone. Phenylethanolamine N-methyltransferase messenger RNA content doubled with restraint but decreased with dexamethasone treatment. As in experiment 1, adrenal catecholamine content increased significantly with dexamethasone treatment. We conclude that without stress, when adrenocorticotropic hormone is low, high doses of exogenous dexamethasone stimulate PNMT and catecholamine synthesis, likely independently of adrenal corticosterone concentration. After stress, adrenocorticotropic hormone levels are elevated, and exogenous dexamethasone suppresses endogenous corticosterone and PNMT production. Nonetheless, catecholamines increase, possibly due to direct neural stimulation, which may override the hormonal regulation of epinephrine synthesis during stress.

Prepubertal exposure to commercial formulation of the herbicide glyphosate alters testosterone levels and testicular morphology

Glyphosate is a herbicide widely used to kill weeds both in agricultural and non-agricultural landscapes. Its reproductive toxicity is related to the inhibition of a StAR protein and an aromatase enzyme, which causes an in vitro reduction in testosterone and estradiol synthesis. Studies in vivo about this herbicide effects in prepubertal Wistar rats reproductive development were not performed at this moment. Evaluations included the progression of puberty, body development, the hormonal production of testosterone, estradiol and corticosterone, and the morphology of the testis. Results showed that the herbicide (1) significantly changed the progression of puberty in a dose-dependent manner; (2) reduced the testosterone production, in semineferous tubules' morphology, decreased significantly the epithelium height (P < 0.001; control = 85.8 +/- 2.8 microm; 5 mg/kg = 71.9 +/- 5.3 microm; 50 mg/kg = 69.1 +/- 1.7 microm; 250 mg/kg = 65.2 +/- 1.3 microm) and increased the luminal diameter (P < 0.01; control = 94.0 +/- 5.7 microm; 5 mg/kg = 116.6 +/- 6.6 microm; 50 mg/kg = 114.3 +/- 3.1 microm; 250 mg/kg = 130.3 +/- 4.8 microm); (4) no difference in tubular diameter was observed; and (5) relative to the controls, no differences in serum corticosterone or estradiol levels were detected, but the concentrations of testosterone serum were lower in all treated groups (P < 0.001; control = 154.5 +/- 12.9 ng/dL; 5 mg/kg = 108.6 +/- 19.6 ng/dL; 50 mg/dL = 84.5 +/- 12.2 ng/dL; 250 mg/kg = 76.9 +/- 14.2 ng/dL). These results suggest that commercial formulation of glyphosate is a potent endocrine disruptor in vivo, causing disturbances in the reproductive development of rats when the exposure was performed during the puberty period.

Dysregulation of Corticosterone Secretion in Streptozotocin-Diabetic Rats: Modulatory Role of the Adrenocortical Nitrergic System

An increased activity of the hypothalamo-pituitary-adrenal axis resulting in exaggerated glucocorticoid secretion has been repeatedly described in patients with diabetes mellitus and in animal models of this disease. However, it has been pointed out that experimental diabetes is accompanied by a decreased glucocorticoid response to ACTH stimulation. Because previous studies from our laboratory demonstrate the involvement of nitric oxide (NO) in the modulation of corticosterone production, present investigations were designed to evaluate 1) the impact of streptozotocin (STZ)-induced diabetes on the adrenocortical nitrergic system and 2) the role of NO in the modulation of adrenal steroidogenesis in STZ-diabetic rats. Four weeks after STZ injection, increased activity and expression levels of proteins involved in L-arginine transport and in NO synthesis were detected, and increased levels of thiobarbituric acid reactive species, carbonyl adducts, and nitrotyrosine-modified proteins were measured in the adrenocortical tissue of hyperglycemic rats. An impaired corticosterone response to ACTH was evident both in vivo and in adrenocortical cells isolated from STZ-treated animals. Inhibition of NO synthase activity resulted in higher corticosterone generation in adrenal tissue from STZ-treated rats. Moreover, a stronger inhibition of steroid output from adrenal cells by a NO donor was observed in adrenocortical Y1 cells previously subjected to high glucose (30 mM) treatment. In summary, results presented herein indicate an inhibitory effect of endogenously generated NO on steroid production, probably potentiated by hyperglycemia-induced oxidative stress, in the adrenal cortex of STZ-treated rats.

Calcitonin directly increases adrenocorticotropic hormone-stimulated corticosterone production in the hen adrenal gland

The present study was performed to elucidate whether the receptor for calcitonin (CT) exists in the adrenocortical cells of hens and to determine the effect of CT on adrenocorticotropic hormone (ACTH)-stimulated corticosterone production in its cell. The binding site of CT in the membrane fraction of the adrenal gland in hens was determined using a [125 I]CT binding assay system. The binding properties in the adrenal gland satisfied the criteria of a receptor-ligand interaction in terms of specificity, reversibility, and saturation. When the cortical cells were incubated in vitro with chicken ACTH in the presence of CT, greater corticosterone production was observed. The result suggested that CT acts directly on the adrenocortical cells via its receptor binding and increases responsiveness of ACTH on corticosterone production in the laying hen.

Influence of age on behavioral, immune and endocrine responses to the T‐cell superantigen staphylococcal enterotoxin A

Aged subjects are more vulnerable to administration of the endotoxin lipopolysaccharide, but research on age-associated sensitivity to other immune stimulants has been limited. The current study examined the effects of administering the superantigen, staphylococcal enterotoxin A (SEA), to young (4-month-old) and aged (20-month-old) male C57BL/6J mice on consumption of a novel liquid, cytokine production, corticosterone levels, and expression of central mRNA levels of cytokines and corticotropin-releasing hormone. SEA produced exaggerated hypophagia in aged mice, as they showed decreased consumption that persisted for 24 h. SEA increased hypothalamic mRNA levels of interleukin-1beta in the aged, but not the young, mice 2 h after administration. No differences in cytokine expression were observed 24 h after SEA. Both age groups showed increased plasma corticosterone levels 2 h after SEA administration. However, 24 h after SEA exposure the aged, but not the young, mice showed an augmented corticosterone response to the consumption test. Collectively, these data show that aging may exacerbate the behavioral and neuroinflammatory response to superantigen exposure. Further, the present study suggests that immune activation may result in delayed alterations in stress-induced corticosterone production in aged subjects.

Propolis effects on pro-inflammatory cytokine production and Toll-like receptor 2 and 4 expression in stressed mice

Introduction Propolis is a beehive product and its immunomodulatory action has been well documented; however, little is known concerning its activity on the immune system of stressed mice. This work investigated a possible role of propolis against the immunosuppressive effects induced by stress in mice, assessing the pro-inflammatory cytokine (IL-1β and IL-6) production and Toll-like receptor (TLR-2 and TLR-4) expression by spleen cells. Methods BALB/c mice were divided into 3 groups: G1 was considered control; G2 was submitted to restraint stress for 3 days, and G3 was treated with propolis and immediately submitted to stress. After sacrifice, spleens were removed and TLR-2 and TLR-4 gene expression was analyzed, as well as the pro-inflammatory cytokine production. Serum corticosterone levels were determined by radioimmunoassay as a stress indicator. Results Stressed mice, treated or not with propolis, produced higher corticosterone levels, whereas IL-1β and IL-6 production was inhibited. TLR-2 and TLR-4 expression was inhibited in stressed mice, while propolis exerted an immunorestorative role in TLR-4 expression. The immunosuppressive effects on IL-1β and IL-6 production and on TLR expression by stressed mice might have occurred due to a higher corticosterone production during stress. Conclusion Propolis treatment did not antagonize the inhibitory effects on pro-inflammatory cytokine production, however it restored at least partially TLR2 mRNA expression and counteracted the inhibition on TLR-4 expression in stressed animals, contributing to the recognition of microorganisms during stressful conditions.