Pituitary Proopiomelanocortin Research Articles

Dysregulated Hypothalamic-Pituitary-Adrenal Axis Is Associated With Increased Inflammation and Worse Outcomes After Ischemic Stroke in Diabetic Mice.

Diabetic patients have larger infarcts, worse neurological deficits, and higher mortality rate after an ischemic stroke. Evidence shows that in diabetes, the hypothalamic–pituitary–adrenal (HPA) axis was dysregulated and levels of cortisol increased. Based on the role of the HPA axis in immunity, we hypothesized that diabetes-dysregulated stress response exacerbates stroke outcomes via regulation of inflammation. To test this hypothesis, we assessed the regulation of the HPA axis in diabetic mice before and after stroke and determined its relevance in the regulation of post-stroke injury and inflammation. Diabetes was induced in C57BL/6 mice by feeding a high-fat diet and intraperitoneal injection of streptozotocin (STZ), and then the mice were subjected to 30 min of middle cerebral artery occlusion (MCAO). Infarct volume and neurological scores were measured in the ischemic mice. The inflammatory cytokine and chemokine levels were also determined in the ischemic brain. To assess the effect of diabetes on the stroke-modulated HPA axis, we measured the expression of components in the HPA axis including corticotropin-releasing hormone (CRH) in the hypothalamus, proopiomelanocortin (POMC) in the pituitary, and plasma adrenocorticotropic hormone (ACTH) and corticosterone. Diabetic mice had larger infarcts and worse neurological scores after stroke. The exacerbated stroke outcomes in diabetic mice were accompanied by the upregulated expression of inflammatory factors (including IL-1β, TNF-α, IL-6, CCR2, and MCP-1) in the ischemic brain. We also confirmed increased levels of hypothalamic CRH, pituitary POMC, and plasma corticosterone in diabetic mice before and after stroke, suggesting the hyper-activated HPA axis in diabetic conditions. Finally, we confirmed that post-stroke treatment of metyrapone (an inhibitor of glucocorticoid synthesis) reduced IL-6 expression and the infarct size in the ischemic brain of diabetic mice. These results elucidate the mechanisms in which the HPA axis in diabetes exacerbates ischemic stroke. Maintaining an optimal level of the stress response by regulating the HPA axis may be an effective approach to improving stroke outcomes in patients with diabetes.

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

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

Characterization of the Interrenal Gland and Sexual Traits Development in cyp17a2-Deficient Zebrafish.

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2-null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2-deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a (pomca) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2-deficient fish. Intriguingly, the cyp17a2-deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2-deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2-/- females and cyp17a2-/- males, respectively. By examining the ovaries development of cyp17a2-deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2-deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2-deficient females.

The co-chaperone FKBP51 modulates HPA axis activity and age-related maladaptation of the stress system in pituitary proopiomelanocortin cells

Glucocorticoid (GC)-mediated negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis, the body’s physiological stress response system, is tightly regulated and essential for appropriate termination of this hormonal cascade. Disturbed regulation and maladaptive response of this axis are fundamental components of multiple stress-induced psychiatric and metabolic diseases and aging. The co-chaperone FK506 binding protein 51 (FKBP51) is a negative regulator of the GC receptor (GR), is highly stress responsive, and its polymorphisms have been repeatedly associated with stress-related disorders and dysfunctions in humans and rodents. Proopiomelanocortin (Pomc)-expressing corticotropes in the anterior pituitary gland are one of the key cell populations of this closed-loop GC-dependent negative feedback regulation of the HPA axis in the periphery. However, the cell type-specific role of FKBP51 in anterior pituitary corticotrope POMC cells and its impact on age-related HPA axis disturbances are yet to be elucidated. Here, using a combination of endogenous knockout and viral rescue, we show that male mice lacking FKBP51 in Pomc-expressing cells exhibit enhanced GR-mediated negative feedback and are protected from age-related disruption of their diurnal corticosterone (CORT) rhythm. Our study highlights the complexity of tissue- and cell type-specific, but also cross-tissue effects of FKBP51 in the rodent stress response at different ages and extends our understanding of potential targets for pharmacological intervention in stress- and age-related disorders.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

OR19-06 Sepsis-Induced Critical Illness in Mice Alters Key Regulators of ACTH Production and Secretion Within the Anterior Pituitary Gland

IntroductionCritical illness is hallmarked by high plasma cortisol without elevated ACTH. This is partly explained by low cortisol-binding proteins and reduced binding affinity and by suppressed cortisol metabolism, 3 peripheral drivers of increased free cortisol (corticosterone (CORT) in rodents).1 We hypothesized that lack of elevated plasma ACTH is explained by CORT-induced feed-back inhibition either due to suppressed CRH or AVP pituitary signaling or to direct glucocorticoid receptor (GR)-mediated action within corticotrophs. To test this hypothesis, we documented alterations in ACTH precursors and key enzymes controlling ACTH maturation and secretion in a mouse model of sepsis-induced critical illness.MethodsC57Bl/6 mice were randomly allocated to a healthy control group or to 4 critically ill groups sacrificed after increasing illness duration (30 hours (H), 3 days (D), 5D or 7D). Critical illness was induced by sepsis brought about by cecal-ligation and puncture followed by fluid-resuscitation and antibiotics treatment. The study was continued until 15 surviving animals per time cohort were reached (n=120).We quantified pituitary pro-opiomelanocortin (POMC) gene/protein expression and POMC plasma concentrations, pituitary POMC intracellular trafficking and cleavage via intracellular POMC sorting/trafficking receptor Carboxypeptidase E (CPE) and prohormone convertase 1 (PC1/3) gene/protein expression. Gene expression of Annexin A1, an inhibitor of mature ACTH secretion, was quantified as marker of GR-mediated CORT-induced feedback inhibition at corticotroph level.ResultsPlasma CORT concentrations were median 3-fold increased during critical illness (p<0.001 for all time cohorts) in the face of normal (for 30H, 3D and 5D cohorts) to low (7D time cohort; p=0.01) plasma ACTH concentrations. Plasma POMC concentrations were higher in critically ill than in control mice (p=0.05). POMC gene expression (but not protein, P=0.8) was a median 55% higher in critically ill mice than in controls (p<0.05 for all time cohorts). In contrast, pituitary mature ACTH protein concentration was median 61% lower in critically ill than in control mice (p<0.01). CPE gene expression was only increased in 30H time cohort (p<0.001). PC1/3 gene and protein expression were positively correlated (R2 0.1; p=0.001) and were reduced (by 37% and 43%, respectively) during the entire course of critical illness (p<0.01). Annexin A1 gene expression was increased during critical illness (p<0.05 for all time cohorts).ConclusionSuppressed CRH or AVP signaling and GR-mediated action within corticotrophs explained lack of elevated plasma ACTH in critical illness, as indicated by impaired POMC processing and ACTH maturation. However, increased POMC gene expression suggests ongoing corticotroph activation, the driver of which needs to be identified. 1. Teblick A et al. Nat Rev Endocrinol 2019

The selective glucocorticoid receptor antagonist CORT125281 has tissue-specific activity.

Glucocorticoids mediate numerous essential processes in the human body via binding to the glucocorticoid receptor (GR). Excessive GR signaling can cause disease, and GR antagonists can be used to treat many symptoms of glucocorticoid-induced pathology. The purpose of this study was to characterize the tissue-specific properties of the selective GR antagonist CORT125281. We evaluated the antagonistic effects of CORT125281 upon acute and subchronic corticosterone exposure in mice. In the acute corticosterone setting, hypothalamus-pituitary-adrenal-axis activity was investigated by measurement of basal- and stress-induced corticosterone levels, adrenocorticotropic hormone levels and pituitary proopiomelanocortin expression. GR signaling was evaluated by RT-PCR analysis of GR-responsive transcripts in liver, muscle, brown adipose tissue (BAT), white adipose tissue (WAT) and hippocampus. Pretreatment with a high dose of CORT125281 antagonized GR activity in a tissue-dependent manner. We observed complete inhibition of GR-induced target gene expression in the liver, partial blockade in muscle and BAT and no antagonism in WAT and hippocampus. Tissue distribution only partially explained the lack of effective antagonism. CORT125281 treatment did not disinhibit the hypothalamus-pituitary-adrenal neuroendocrine axis. In the subchronic corticosterone setting, CORT125281 partially prevented corticosterone-induced hyperinsulinemia, but not hyperlipidemia and immune suppression. In conclusion, CORT125281 antagonizes GR transcriptional activity in a tissue-dependent manner and improves corticosterone-induced hyperinsulinemia. Tailored dosing of CORT125281 may allow tissue-specific inhibition of GR transcriptional activity.

Structural basis of NR4A1 bound to the human pituitary proopiomelanocortin gene promoter

The nuclear receptor NR4A subfamily (NR4A1/NGFI-B, NR4A2/Nurr1 and NR4A3/NOR-1) can recognize different classes of DNA response elements either as a monomer, homodimer, or heterodimer. In this study, we determined the structure of the NR4A1 DNA-binding domain (NR4A1-DBD) bound to natural Nur-responsive elements (NurREs) in the promoter region of the pituitary proopiomelanocortin (POMC) gene (NurREPOMC) at 3.12 Å resolution. The NR4A1-DBD molecules bound independently to this element in our structure. The N-terminal helix H1 forms specific contacts with major groove, and C-terminal extension interact extensively with minor groove. Moreover our modelling structure of NR4A1 large fragment complexed with NurREPOMC indicated that ligand binding domain of NR4A might form dimer interactions to help recognize DNA. Overall, our analyses provide a molecular basis for DNA binding of NR4A proteins as a homodimer and novel insight into the molecular functions of NR4A modulation of gene expression.

Ketamine and Etomidate Down-regulate the Hypothalamic-Pituitary-Adrenal Axis in an Endotoxemic Mouse Model.

We compared the effects of etomidate and ketamine on the hypothalamic-pituitary-adrenal axis during sepsis. Mice (n = 5/group) were injected intraperitoneally with lipopolysaccharide (10 mg/kg) and 6 h later randomized to receive ketamine (100 mg/kg), etomidate (30 mg/kg), or saline. At two time points (12 and 48 h), messenger RNA levels of hypothalamic corticotropin-releasing hormone, pituitary proopiomelanocortin, and four adrenal enzymes (P450 side-chain cleavage, 3β-hydroxysteroid deshydrogenase, 21-hydroxylase, and 11β-hydroxylase) were measured by in situ hybridization (results are presented as optical density), and plasma levels of corticosterone and adrenocorticotropin hormones were measured by enzyme-linked immunosorbent assay (mean ± SD). At 12 h, lipopolysaccharide induced an overexpression of corticotropin-releasing hormone (32 ± 5 vs. 18 ± 6, P < 0.01), proopiomelanocortin (21 ± 3 vs. 8 ± 0.9, P < 0.0001), P450 side-chain cleavage (32 ± 4 vs. 23 ± 10, P < 0.05), 21-hydroxylase (17 ± 5 vs. 12 ± 2, P < 0.05), and 11β-hydroxylase (11 ± 4 vs. 6 ± 0.5, P = 0.001), and an elevation of corticosterone (642 ± 165 vs. 98.3 ± 63 ng/ml, P < 0.0001). Etomidate and ketamine reduced P450 side-chain cleavage (19 ± 7 and 19 ± 3 vs. 32 ± 4, P < 0.01), 21-hydroxylase (8 ± 0.8 and 8 ± 1 vs. 17 ± 5, P < 0.001), 11β-hydroxylase (4 ± 0.5 and 7 ± 1 vs. 11 ± 4, P < 0.001 and P < 0.05), and corticosterone (413 ± 189 and 260 ± 161 vs. 642 ± 165 ng/ml, P < 0.05 and P < 0.01). Ketamine also inhibited adrenocorticotropin hormone production (2.5 ± 3.6 vs. 36 ± 15 pg/ml, P < 0.05). At 48 h, all four adrenal enzymes were down-regulated by lipopolysaccharide administration with corticosterone levels similar to the control group. Ketamine and etomidate did not modify corticosterone plasma levels. Our endotoxemic model induces an initial activation of the hypothalamic-pituitary-adrenal axis, followed by a secondary inhibition of adrenal steroidogenesis processes. Ketamine and etomidate inhibit the enzyme expression and activity of the adrenal gland at the early stage.

A decline in female baboon hypothalamo-pituitary-adrenal axis activity anticipates aging.

Stressors that disrupt homeostasis advance aging. Glucocorticoids regulate multiple processes that determine the aging trajectory. Debate exists regarding life-course circulating glucocorticoid concentrations. Rodent and nonhuman primate studies indicate circulating glucocorticoids fall from early life. We measured fasting morning cortisol in 24 female baboons (6-21 years, human equivalent ~18-70). We also quantified hypothalamic paraventricular nuclear (PVN) arginine vasopressin (AVP), corticotropin-releasing hormone, steroid receptors, and pituitary proopiomelanocortin immunohistochemically in 14 of these females at 6-13 years. We identified significant age-related 1) linear fall in cortisol and PVN AVP from as early as 6 years; 2) increased PVN glucocorticoid and mineralocorticoid receptors; 3) increased PVN 11β-hydroxysteroid dehydrogenase 1 and 2, regulators of local cortisol production, and 4) decreased pituitary proopiomelanocortin. Our data identify increased age-related negative feedback and local PVN cortisol production as potential mechanisms decreasing PVN drive to hypothalamo-pituitary-adrenal axis activity that result in the age-related circulating cortisol fall. Further studies are needed to determine whether the cortisol fall 1) causes aging, 2) protects by slowing aging, or 3) is an epiphenomenon unrelated to aging processes. We conclude that aging processes are best studied by linear life-course analysis beginning early in life.

Anxiogenic-like effects of fluoxetine render adult male rats vulnerable to the effects of a novel stress

Fluoxetine (FLX) has paradoxical anxiogenic-like effects during the acute phase of treatment. In adolescent (35d-old) male rats, the stress-like effects induced by short-term (3d–4d) FLX treatment appear to involve up-regulation of paraventricular nucleus (PVN) arginine vasopressin (AVP) mRNA. However, studies on FLX-induced anxiety-like effects in adult rodents are inconclusive. Herein, we sought to study the response of adult male rats (60–65d-old) to a similar FLX treatment, also investigating how the stressful component, inherent to our experimental conditions, contributed to the responses. We show that FLX acutely increased plasma corticosterone concentrations while it attenuated the stress-induced-hyperthermia (SIH) as well as it reduced (≈40%) basal POMC mRNA expression in the arcuate nucleus (ARC). However, FLX did not alter the basal expression of PVN-corticotrophin-releasing hormone (CRH), anterior pituitary-pro-opiomelanocortin (POMC) and raphe nucleusserotonin transporter (SERT). Nonetheless, some regressions point towards the plausibility that FLX activated the hypothalamic-pituitary-adrenal (HPA). The behavioral study revealed that FLX acutely increased emotional reactivity in the holeboard, effect followed by a body weight loss of ≈2.5% after 24h. Interestingly, i.p. injection with vehicle did not have behavioral effects, furthermore, after experiencing the stressful component of the holeboard, the rats kept eating and gaining weight as normal. By contrast, the stress-naïve rats reduced food intake and gained less weight although maintaining a positive energy state. Therefore, on one hand, repetition of a mild stressor would unchain compensatory mechanisms to restore energy homeostasis after stress increasing the resiliency to novel stressors. On the other hand, FLX might render stressed adult rats vulnerable to novel stressors through the emergence of counter-regulatory changes, involving HPA axis activation and diminished sympathetic output may be due to reduced melanocortin signaling. Therefore, complex interactions between hypothalamic CRH and POMC might be determining the adaptive nature of the response of adult male rats to FLX and/or stress.

Estrogen Suppresses Interaction of Melanocortin 2 Receptor and Its Accessory Protein in the Primate Fetal Adrenal Cortex.

We have shown that fetal adrenal fetal zone (FZ) volume and serum dehydroepiandrosterone sulfate (DHAS) levels were increased, whereas definitive and transitional zone (DZ/TZ) volume was unaltered, in baboons in which estrogen levels were suppressed by the administration of the aromatase inhibitor letrozole. The interaction of the melanocortin 2 receptor (MC2R) with its accessory protein (MRAP) is essential for trafficking MC2R to the adrenal cell surface for binding to ACTH. The present study determined whether the estrogen-dependent regulation of fetal adrenocortical development is mediated by ACTH and/or expression/interaction of MC2R and MRAP. Fetal pituitary proopiomelanocortin mRNA and plasma ACTH levels and fetal adrenal MC2R-MRAP interaction were assessed in baboons in which estrogen was suppressed/restored by letrozole/letrozole plus estradiol administration during the second half of gestation. Although fetal pituitary proopiomelanocortin and plasma ACTH levels and fetal adrenal MC2R and MRAP protein levels were unaltered, MC2R-MRAP interaction was 2-fold greater (P < .05) in the DZ/TZ in letrozole-treated baboons than in untreated animals and restored by letrozole plus estradiol treatment. We propose that the increasing levels of estradiol with advancing pregnancy suppress interaction of MC2R with MRAP, thereby diminishing MC2R movement to the cell membrane in the DZ/TZ. This would be expected to reduce progenitor cell proliferation in the DZ and migration to the FZ, thereby restraining FZ growth and DHAS production to maintain fetal adrenal DHAS and placental estradiol levels in a physiological range late in gestation.

Improvement of kidney yang syndrome by icariin through regulating hypothalamus-pituitary-adrenal axis.

To investigate whether Epimedium brevicornu Maxim (EB) and icariin could exert their protective effects on hydrocortisone induced (HCI) rats by regulating the hypothalamus-pituitary-adrenal (HPA) axis and endocrine system and the possible mechanism. Male 10-week-old Sprague Dawley (SD) rats were allotted to 6 groups (A-F) with 12 each, group A was injected normal saline (NS) 3 mL/kg day intraperitoneally, group A and B were given NS 6 mL/kg day by gastrogavage, group B-F were injected hydrocortisone 15 mg/kg intraperitoneally, group C and D were given EB 8 or 5 g/(kg day) by gastrogavage, group E and F were given icariin 25 or 50 mg/(kg day) by gastrogavage. Gene expressions of hypothalamus corticotropin releasing hormone (CRH) and pituitary proopiomelanocortin (POMC) were detected by reverse transcription-polymerase chain reaction (RT-PCR), and protein of pituitary POMC by Western-blot. The serum T4, testosterone, cortisol and POMC mRNA expression were increased after treatment with EB or icariin in HCI rats, the serum CRH and the hypothalamus CRH mRNA expression released from hypothalamus corticotropin decreased compared with group B (P<0.05).The treatment with only icariin increased serum adrenocorticotropic hormone (ACTH) compared with group B (P<0.05). EB and icariin might be therapeutically beneficial in the treatment of HCI rats through attuning the HPA axis and endocrine system which was involved in the release of CRH in hypothalamic, and the production of POMC-derived peptide ACTH in anterior pituitary, the secretion of corticosteroids in adrenal cortex.

Expression and regulation of neuromedin B in pituitary corticotrophs of male melanocortin 2 receptor-deficient mice.

The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R(-/-) mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R(-/-) mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R(-/-) mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R(-/-) than in the wild-type (WT) mice. We detected NMB expression in the MC2R(-/-) pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R(-/-) mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Early-Life Stress Reduces DNA Methylation of the Pomc Gene in Male Mice

Early-life stress (ELS) increases the vulnerability thresholds for stress-related diseases such as major depression and anxiety by inducing alterations in the structure and function of neural circuits and endocrine pathways. We previously demonstrated the contribution of epigenetic mechanisms to the long-term programming of the hypothalamo-pituitary-adrenal axis activity following ELS exposure in male mice. Here, ELS comprising daily separation of pups from their dams on postnatal days 1-10 was observed to up-regulate the expression of the pituitary proopiomelanocortin (Pomc) gene; POMC serves as a prohormone for ACTH, a key mediator of the adrenocortical response to stress. Detailed analysis revealed that the increase in Pomc mRNA levels results from a reduction in DNA methylation at a critical regulatory region of the Pomc gene; interestingly, this change occurs with some delay after ELS and persists for up to 1 year. Using a Pomc-expressing pituitary cell line (AtT20), we confirmed a role for DNA methylation in restraining Pomc expression under resting conditions: specifically, we show that CpG site-specific methylation of the Pomc promoter represses Pomc mRNA transcription. Further, we show high-affinity binding of methyl-CpG binding protein-2 to the distal promoter of Pomc, suggesting that methyl-CpG binding protein-2 acts in association with the chromatin modifiers histone deacetylase 2 and DNA methyltransferase 1 to repress Pomc gene expression. Collectively, these experiments contribute to our understanding of the mechanisms through which environmental cues are translated into stable changes ("cellular memory") in neuroendocrine cells.

Fish Oil Alleviates Activation of the Hypothalamic-Pituitary-Adrenal Axis Associated with Inhibition of TLR4 and NOD Signaling Pathways in Weaned Piglets after a Lipopolysaccharide Challenge

Long-chain n–3 (ω-3) polyunsaturated fatty acids exert beneficial effects in neuroendocrine dysfunctions in animal models and clinical trials. However, the mechanism(s) underlying the beneficial effects remains to be elucidated. We hypothesized that dietary treatment with fish oil (FO) could mitigate LPS-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis through inhibition of Toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling pathways. Twenty-four weaned pigs were used in a 2 × 2 factorial design, and the main factors consisted of diet (5% corn oil vs. 5% FO) and immunological challenge (saline vs. LPS). After 21 d of dietary treatment with 5% corn oil or FO diets, pigs were treated with saline or LPS. Blood samples were collected at 0 (preinjection), 2, and 4 h postinjection, and then pigs were humanely killed by intravenous injection of 40 mg/kg body weight sodium pentobarbital for tissue sample collection. FO led to enrichment of eicosapentaenoic acid and docosahexaenoic acid and total n–3 polyunsaturated fatty acids in hypothalamus, pituitary gland, adrenal gland, spleen, and thymus. FO decreased plasma adrenocorticotrophin and cortisol concentrations as well as mRNA expressions of hypothalamic corticotropin releasing hormone and pituitary proopiomelanocortin. FO also reduced mRNA expression of tumor necrosis factor-α in hypothalamus, adrenal gland, spleen, and thymus, and of cyclooxygenase 2 in hypothalamus. Moreover, FO downregulated the mRNA expressions of Toll-like receptor 4 (TLR4) and its downstream molecules, including cluster differentiation factor 14, myeloid differentiation factor 2, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase 1, tumor necrosis factor-α receptor-associated factor 6, and nuclear factor kappa-light-chain-enhancer of activated B cells p65, and also decreased the mRNA expressions of nucleotide-binding oligomerization domain 1, nucleotide-binding oligomerization domain 2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2. These results suggested that FO attenuates the activation of the HPA axis induced by LPS challenge. The beneficial effects of FO on the HPA axis may be associated with decreasing the production of brain or peripheral proinflammatory cytokines through inhibition of TLR4 and nucleotide-binding oligomerization domain protein signaling pathways.

Acute selective ablation of rat insulin promoter-expressing (RIP HER ) neurons defines their orexigenic nature

Rat insulin promoter (RIP)-expressing neurons in the hypothalamus control body weight and energy homeostasis. However, genetic approaches to study the role of these neurons have been limited by the fact that RIP expression is predominantly found in pancreatic β-cells, which impedes selective targeting of neurons. To define the function of hypothalamic RIP-expressing neurons, we set out to acutely and selectively eliminate them via diphtheria toxin-mediated ablation. Therefore, the diphtheria toxin receptor transgene was specifically expressed upon RIP-specific Cre recombination using a RIP-Cre line first described by Herrera (RIP(HER)-Cre) [Herrera PL (2000) Development 127:2317-2322]. Using proopiomelanocortin-expressing cells located in the arcuate nucleus of the hypothalamus and in the pituitary gland as a model, we established a unique protocol of intracerebroventricular application of diphtheria toxin to efficiently ablate hypothalamic cells with no concomitant effect on pituitary proopiomelanocortin-expressing corticotrophs in the mouse. Using this approach to ablate RIP(HER) neurons in the brain, but not in the pancreas, resulted in decreased food intake and loss of body weight and fat mass. In addition, ablation of RIP(HER) neurons caused increased c-Fos immunoreactivity of neurons in the paraventricular nucleus (PVN) of the hypothalamus. Moreover, transsynaptic tracing of RIP(HER) neurons revealed labeling of neurons located in the PVN and dorsomedial hypothalamic nucleus. Thus, our experiments indicate that RIP(HER) neurons inhibit anorexigenic neurons in the PVN, revealing a basic orexigenic nature of these cells.

The impact of maternal synthetic glucocorticoid administration in late pregnancy on fetal and early neonatal hypothalamic-pituitary-adrenal axes regulatory genes is dependent upon dose and gestational age at exposure.

In this study, we determined the gene and/or protein expression of hypothalamic-pituitary-adrenal (HPA) axis regulatory molecules following synthetic glucocorticoid exposures. Pregnant sheep received intramuscular saline or betamethasone (BET) injections at 104 (BET-1), 104 and 111(BET-2) or 104, 111 and 118 (BET-3) days of gestation (dG). Samples were collected at numerous time-points between 75 dG and 12 weeks postnatal age. In the BET-3 treatment group, fetal plasma cortisol levels were lower at 145 dG than controls and gestational length was lengthened significantly. The cortisol:adrenocorticotropic hormone (ACTH) ratio in fetal plasma of control and BET-3 fetuses rose significantly between132 and 145 dG, and remained elevated in lambs at 6 and 12 weeks of age; this rise was truncated at day 145 in fetuses of BET-3 treated mothers. After BET treatment, fetal and postnatal pituitary proopiomelanocortin mRNA levels were reduced from 109 dG to 12 weeks postnatal age; pituitary prohormone convertase 1 and 2 mRNA levels were reduced at 145 dG and postnatally; hypothalamic arginine vasopressin mRNA levels were lowered at all time-points, but corticotrophin-releasing hormone mRNA levels were reduced only in postnatal lambs. Maternal BET increased late fetal and/or postnatal adrenal mRNA levels of ACTH receptor and 3β hydroxysteroid dehydrogenase but decreased steroidogenic acute regulatory protein and P450 17-α hydroxylase. The altered mRNA levels of key HPA axis regulatory proteins after maternal BET injections suggests processes that may subserve long-term changes in HPA activity in later life after prenatal exposure to synthetic glucocorticoids.

Plasticity in photoperiodic regulation of adrenal, but not testicular, function in Syrian hamsters

Transfer from long days (LD) to short days (SD) increases aggressive behavior, but it suppresses the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes in male Syrian hamsters. The present study sought to determine whether social instability (group housing from days 1–70, single housing from days 71 to 84, and 10-min social encounters during the light or dark phase on days 82 and 83) could reverse SD-induced quiescence in the aggression-promoting HPA and HPG axes. Controls were housed in stable groups during LD or SD exposure. Euthanasia occurred on day 84 during the light or dark phase (unstable condition) and during the dark phase (stable condition). SD exposure in the unstable condition increased aggression during social pairings, and it elevated circulating corticosterone, cortisol, and adrenocorticotropic hormone (ACTH) concentrations, assessed by RIA, particularly during the dark phase. Although anterior pituitary pro-opiomelanocortin (POMC) immunoreactivity was unaltered by these experimental conditions, SD and the dark phase during social instability elevated POMC mRNA levels, assessed by solution hybridization assay. In socially stable controls, SD exposure increased aggression, assessed by bite marks, reduced cortisol and ACTH, but not corticosterone, secretion, and it reduced anterior pituitary POMC mRNA, but not immunoreactivity, levels. SD exposure in both conditions reduced testicular function, indicated by more than 77% reduction of testis mass. These results suggest that social instability, rather than aggression per se, reversed SD-induced suppression of HPA, but not HPG, function.

The Effects of Dexamethasone Treatment in Early Gestation on Hypothalamic–Pituitary–Adrenal Responses and Gene Expression at 7 Months of Postnatal Age in Sheep

We determined the effects of prenatal dexamethasone administration in early gestation on development of the hypothalamic-pituitary-adrenal (HPA) axis up to 7 months of postnatal age with measurements of hormone levels and gene expression. Plasma adrenocorticotropic hormone and cortisol levels after corticotropin-releasing hormone (CRH)/arginine vasopressin challenge were lower in treatment females than in control females and treatment males. Calculation of cortisol to adrenocorticotropic hormone ratios indicated however that the adrenals of treatment females were more responsive to adrenocorticotropic hormone than control females or treatment males. Effects of treatment and sex dependence at 7 months of age were observed in levels of hypothalamic CRH messenger RNA (mRNA), hypothalamic arginine vasopressin mRNA, pituitary proopiomelanocortin mRNA, pituitary prohormone convertase 1 and prohormone convertase 2, glucocorticoid receptor and mineralocorticoid receptor in the hypothalamus and hippocampus, adrenal adrenocorticotropic hormone receptor, steroidogenic acute regulatory, 3β hydroxysteroid dehydrogenase, and 11β hydroxysteroid dehydrogenase type 2 mRNA. The results indicate that exposure to glucocorticoids in early pregnancy produces persisting and sex-dependent effects on the hypothalamic-pituitary-adrenal axis at 7 months of age.