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Objectives Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterised by isolated glucocorticoid deficiency. Melanocortin receptor 2 (MC2R) mediates the functions of adrenocorticotropic hormone (ACTH) in the adrenal cortex. MC2R accessory protein (MRAP) is a transmembrane protein involved in the trafficking of MC2R to the cell surface. Mutations in MC2R and MRAP genes cause FGD type 1 and 2. In the present case series, we evaluate the clinical characteristics and long-term follow-up of six cases with FGD due to mutations in MC2R and MRAP. Case presentation Data of six cases with FGD (five with mutations in MC2R and one with a mutation in MRAP) who were being followed at our paediatric endocrine centre was evaluated. Diagnosis of FGD was considered in case of elevated ACTH and inappropriately low cortisol level, and exclusion of other aetiologies. The main presenting complaints were hyperpigmentation and hypoglycaemic convulsion in all cases. During a follow-up period of 26–115 months, one patient with homozygous 560delT mutation in MC2R, one female with G226R mutation in MC2R and one female with IVS3ds+1delG mutation in MRAP had a neurodevelopmental delay (NDD), while the other three patients had normal neurodevelopment. Conclusions FGD patients due to MC2R and MRAP mutations with early diagnosis and compliance to the hydrocortisone therapy had normal neurodevelopment, while delay in diagnosis and poor compliance was associated with severe hypoglycaemic convulsions and subsequent complications NDD.

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing’s syndrome. Adrenal specimens from six patients with Cushing’s disease were incubated with 10 nM–1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10–100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing’s disease.

Background The Period Circadian Regulator 2 (Per2) gene is important for the modulation of circadian rhythms that influence biological processes. Circadian control of the hypothalamus-pituitary-adrenal (HPA) axis is critical for regulation of hormones involved in the stress response. Dysregulation of the HPA axis is associated with neuropsychiatric disorders. Therefore, it is important to understand how disruption of the circadian rhythm alters the HPA axis. One way to address this question is to delete a gene involved in regulating a central circadian gene such as Per2 in an animal model and to determine how this deletion may affect the HPA axis and behaviors that are altered when the HPA axis is dysregulated. To study this, corticosterone (CORT) levels were measured through the transition from light (inactive phase) to dark (active phase). Additionally, CORT levels as well as pituitary and adrenal mRNA expression were measured following a mild restraint stress. Mice were tested for depressive-like behaviors (forced swim test (FST)), acoustic startle response (ASR), and pre-pulse inhibition (PPI). Results The present results showed that Per2 knockout impacted CORT levels, mRNA expression, depressive-like behaviors, ASR and PPI. Unlike wild-type (WT) mice, Per2 knockout (Per2) mice showed no diurnal rise in CORT levels at the onset of the dark cycle. Per2−/− mice had enhanced CORT levels and adrenal melanocortin receptor 2 (Mc2R) mRNA expression following restraint. There were no changes in expression of any other pituitary or adrenal gene. In the FST, Per2−/− mice spent more time floating (less time struggling) than WT mice, suggesting increased depressive-like behaviors. Per2−/− mice had deficits in ASR and PPI startle responses compared to WT mice. Conclusions In summary, these findings showed that disruption of the circadian system via Per2 gene deletion dysregulated the HPA stress axis and is subsequently correlated with increased depressive-like behaviors and deficits in startle response.

IntroductionAdrenocortical hyperplasia and adrenal rest tumor (ART) formation are common in congenital adrenal hyperplasia (CAH). Although driven by excessive corticotropin, much is unknown regarding the morphology and transformation of these tissues. Our study objective was to characterize CAH-affected adrenals and ART and compare with control adrenal and gonadal tissues.Patients/MethodsCAH adrenals, ART and control tissues were analyzed by histology, immunohistochemistry, and transcriptome sequencing. We investigated protein expression of the ACTH receptor (MC2R), steroidogenic (CYP11B2, CYP11B1, CYB5A) and immune (CD20, CD3, CD68) biomarkers, and delta-like 1 homolog (DLK1), a membrane bound protein broadly expressed in fetal and many endocrine cells. RNA was isolated and gene expression was analyzed by RNA sequencing (RNA-seq) followed by principle component, and unsupervised clustering analyses.ResultsBased on immunohistochemistry, CAH adrenals and ART demonstrated increased zona reticularis (ZR)-like CYB5A expression, compared to CYP11B1, and CYP11B2, markers of zona fasciculata and zona glomerulosa respectively. CYP11B2 was mostly absent in CAH adrenals and absent in ART. DLK1 was present in CAH adrenal, ART, and also control adrenal and testis, but was absent in control ovary. Increased expression of adrenocortical marker MC2R, was observed in CAH adrenals compared to control adrenal. Unlike control tissues, significant nodular lymphocytic infiltration was observed in CAH adrenals and ART, with CD20 (B-cell), CD3 (T-cell) and CD68 (macrophage/monocyte) markers of inflammation. RNA-seq data revealed co-expression of adrenal MC2R, and testis-specific INSL3, HSD17B3 in testicular ART indicating the presence of both gonadal and adrenal features, and high expression of DLK1 in ART, CAH adrenals and control adrenal. Principal component analysis indicated that the ART transcriptome was more similar to CAH adrenals and least similar to control testis tissue.ConclusionsCAH-affected adrenal glands and ART have similar expression profiles and morphology, demonstrating increased CYB5A with ZR characteristics and lymphocytic infiltration, suggesting a common origin that is similarly affected by the abnormal hormonal milieu. Immune system modulators may play a role in tumor formation of CAH.

Melanocortin-2 receptor accessory protein (MRAP) has an unusual dual topology and influences the expression, localisation, signalling and internalisation of the melanocortin receptor 2 (MC(2)); the adrenocorticotropic hormone (ACTH) receptor. Mutations in MRAP are associated with familial glucocorticoid deficiency type-2 and evidence is emerging of the importance of MRAP in adrenal development and ACTH signalling. Human MRAP has two functional splice variants: MRAP-α and MRAP-β, unlike MRAP-β, MRAP-α has little expression in brain but is highly expressed in ovary. MRAP2, identified through whole human genome sequence analysis, has approximately 40% sequence homology to MRAP. MRAP2 facilitates MC2 localisation to the cell surface but not ACTH signalling. MRAP and MRAP2 have been found to regulate the surface expression and signalling of all melanocortin receptors (MC(1–5)). Additionally, MRAP2 moderates the signalling of the G-protein coupled receptors (GCPRs): orexin, prokineticin and GHSR1a; the ghrelin receptor. Whilst MRAP appears to be mainly involved in glucocorticoid synthesis, an important role is emerging for MRAP2 in regulating appetite and energy homeostasis. Transgenic models indicate the importance of MRAP in adrenal gland formation. Like MC3R and MC4R knockout mice, MRAP2 knockout mice have an obese phenotype. In vitro studies indicate that MRAP2 enhances the MC3 and MC4 response to the agonist αMSH, which, like ACTH, is produced through precursor polypeptide proopiomelanocortin (POMC) cleavage. Analysis of cohorts of individuals with obesity have revealed several MRAP2 genetic variants with loss of function mutations which are causative of monogenic hyperphagic obesity with hyperglycaemia and hypertension. MRAP2 may also be associated with female infertility. This review summarises current knowledge of MRAP and MRAP2, their influence on GPCR signalling, and focusses on pathophysiology, particularly familial glucocorticoid deficiency type-2 and obesity.

Background: The mechanisms leading to elevated aldosterone synthesis in aldosterone-producing adenomas (APAs) remain an area of active research. Aldosterone-driver somatic gene mutations that allow inappropriate intracellular calcium entrance have been identified in most APAs. Cell-based studies of such mutations indicate that responses to physiologic stimuli, such as angiotensin II or ACTH, are increased. Little is known, however, regarding possible variations in response to hormonal stimuli between APAs with different aldosterone-driver mutations. Herein, we analyzed the transcript expression of the ACTH receptor (MC2R), the melanocortin 2 receptor accessory protein (MRAP) and the type 1 angiotensin II receptor (AGTR1) in APAs with known aldosterone-driver somatic mutations. Methods: RNA was isolated from normal adrenal glands (n=8), and from APAs with mutations in: KCNJ5 (n=14), ATP1A1 (n=14), CACNA1D (n=14), and ATP2B3 (n=5). The gene expressions of MC2R, MRAP, AGTR1 and aldosterone synthase (CYP11B2) were quantified using qPCR and normalized to β-actin. Results: All APA mutation groups had significantly higher transcript levels of CYP11B2, MC2R and AGTR1 as compared to whole normal adrenals. While MRAP and AGTR1 transcripts were comparable between tumor mutation groups, MC2R expression was significantly lower in KCNJ5-mutated APAs compared to other APAs. Overall, CYP11B2 expression demonstrated positive correlations with MC2R (R=0.728, p

Objectives Primary bilateral macronodular adrenal hyperplasia is an uncommon cause of endogenous Cushing's syndrome characterized by the presence of aberrant adrenal expression of ectopic receptors that regulate steroidogenesis by mimicking the events triggered by ACTH receptor activation. Receptors of this type have been described for several hormones. The aim of the study is to detect these receptors in two patients with ACTH-independent hypercortisolism by means of the application of a screening protocol. Design and methods A protocolized study of aberrant receptors was performed including measurements of ACTH, cortisol and other steroids and hormones. Upright posture test, mixed food and administration of Gonadotropin-Releasing Hormone (GnRH) were used as stimuli. In both patients, a stimulation test with intravenous ACTH was conducted to determinate the cortical response capacity. The study was carried out in three separate days. Results The first patient, who had a hypergonadotropic hypogonadism , presented anomalous cortisol response to the GnRH stimulation, with potential medical treatment by the use of exogenous testosterone . In the second case, the patient with clinical Cushing's syndrome presented anomalous cortisol response to standing, whose potential medical treatment would be the use of beta-blockers . Conclusions This etiological variant of ACTH-independent Cushing's syndrome leads to the use of specific pharmacologic therapies in some cases as alternatives to adrenalectomy . The studied cases show the importance of having a high degree of suspicion when diagnosing less frequent types of Cushing's syndrome.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

ACTH, a melanocortin peptide used to treat multiple sclerosis (MS) relapses, acts by stimulating adrenal corticosteroid (CS) production via melanocortin receptor 2 (MC2R), but it may also exert a therapeutic effect independent of CS by stimulating other melanocortin receptors (MCR) distributed in many tissues, including the brain. We reported that oligodendroglia (OL) and oligodendroglial precursor cells (OPC) express MC4R, and that ACTH 1-39 protects OL and OPC in vitro from cell death induced by mechanisms likely involved in white matter damage in MS. This study investigates expression of MC1R, MC2R, MC3R and MC5R in OL and MC4R in OPC using immunocytochemistry with MCR subtype specific antibodies. OL express surface MC1R, MC3R and MC5R, in addition to MC4R. To investigate whether these receptors are functional, we asked if signaling through MCR is involved in ACTH protection of cultured rat OL from apoptosis (staurosporine), or cell death induced by excitotoxicity (glutamate), reactive oxygen species (ROS), or an inflammatory mediator (quinolinic acid). Like ACTH 1-39, MCR subtype specific agonists for MC1R, MC3R, MC4R and MC5R all protected OL from these insults. Conversely, antagonists for MC3R and MC4R blocked ACTH protection of OL. We then investigated the role of MC4R, as a prototype MCR, in protection and proliferation of OPC; MC4R agonists protected OPC and increased their proliferation, while antagonists blocked these effects. Our results demonstrate that MCR on OL and OPC are functional and activate signaling pathways that protect against mechanisms involved in OL damage in MS, suggesting potential beneficial effects in neurologic diseases.

Primary adrenal insufficiency (PAI) is potentially life threatening, but rare. In children, genetic defects prevail whereas adults suffer more often from acquired forms of PAI. The spectrum of genetic defects has increased in recent years with the use of next-generation sequencing methods and now has reached far beyond genetic defects in all known enzymes of adrenal steroidogenesis. Cofactor disorders such as P450 oxidoreductase (POR) deficiency manifesting as a complex form of congenital adrenal hyperplasia with a broad clinical phenotype have come to the fore. In patients with isolated familial glucocorticoid deficiency (FGD), in which no mutations in the genes for the ACTH receptor (MC2R) or its accessory protein MRAP have been found, non-classic steroidogenic acute regulatory protein (StAR) andCYP11A1mutations have been described; and more recently novel mutations in genes such as nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TRXR2) involved in the maintenance of the mitochondrial redox potential and generation of NADPH important for steroidogenesis and ROS detoxication have been discovered. In addition, whole exome sequencing approach also solved the genetics of some syndromic forms of PAI including IMAGe syndrome (CDKN1C), Irish traveler syndrome (MCM4), MIRAGE syndrome (SAMD9); and most recently a syndrome combining FGD with steroid-resistant nephrotic syndrome and ichthyosis caused by mutations in the gene for sphingosine-1-phosphate lyase 1 (SGPL1). This review intends do give an update on novel genetic forms of PAI and their suggested mechanism of disease. It also advocates for advanced genetic work-up of PAI (especially in children) to reach a specific diagnosis for better counseling and treatment.

Alopecia areata (AA) is an autoimmune disease of the hair follicle. Keratinocytes of the hair follicle generate an immunosuppressive environment by the local secretion of hormones of the hypothalamic-pituitary-adrenal axis of the skin (skin HPA analog). Our objective was to measure the local production of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and α-melanocyte-stimulating hormone (α-MSH) in the scalp tissue of patients with AA before and after ultraviolet A1 (UVA-1) phototherapy to determine their role in the pathogenesis of AA and the effect of UVA-1 on the AA hormonal environment. This was a retrospective and descriptive study of skin samples from 22 patients with AA before and after UVA-1 treatment. We compared the changes in the local hormonal environment by measuring CRH, ACTH, type 2 melanocortin receptor (ACTH receptor) and α-MSH with immunohistochemical stains. The positivity of MSH was significantly higher (P = .037) in the post-treatment samples compared with the baseline value. ACTH was significantly higher in intensity (P = .032) in the post-treatment samples compared with the initial value. CRH was significantly higher in intensity (P = .013) in baseline samples compared with the final biopsies. The positivity of the ACTH receptor MC2R was not different between the two groups (P = .626). In AA, an interruption in the signalling of CRH could decrease the local concentration of ACTH and MSH, and consequently, the immunosuppressive effect of these hormones. This phenomenon is normalized in the skin treated with UVA-1. A defective signalling system in the cutaneous HPA axis may be involved in the pathogenesis of AA.

In the neuroendocrine system, corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) play important roles in the regulation of the hypothalamic-pituitary-adrenal (HPA) system. Disorders of the HPA system lead to physiological problems, such as Addison's disease and Cushing's syndrome. Therefore, detection of CRH and ACTH is essential for diagnosing disorders related to the HPA system. Herein, receptors of the HPA axis were used to construct a bioelectronic sensor system for the detection of CRH and ACTH. The CRH receptor, corticotropin-releasing hormone receptor 1 (CRHR1), and the ACTH receptor, melanocortin 2 receptor (MC2R), were produced using an Escherichia coli expression system, and were reconstituted using nanodisc (ND) technology. The receptor-embedded NDs were immobilized on a floating electrode of a carbon nanotube field-effect transistor (CNT-FET). The constructed sensors sensitively detected CRH and ACTH to a concentration of 1 fM with high selectivity in real time. Furthermore, the reliable detection of CRH and ACTH in human plasma by the developed sensors demonstrated their potential in clinical and practical applications. These results indicate that CRHR1 and MC2R-based bioelectronic sensors can be applied for rapid and efficient detection of CRH and ACTH.

Corticosteroid-binding globulin (CBG) is synthesized by the liver and secreted into the bloodstream where binds to glucocorticoids. Thus CBG has the role of glucocorticoid transport and free hormone control. In addition, CBG has been detected in some extrahepatic tissues without a known role. CBG-deficient mice show decreased total corticosterone levels with missing of classical sexual dimorphism, increased free corticosterone, higher adrenal gland size and altered HPA axis response to stress. Our aim was to ascertain whether CBG deficiency could affect the endocrine synthetic activity of adrenal gland and if the adrenal gland produces CBG. We determined the expression in adrenal gland of proteins involved in the cholesterol uptake and its transport to mitochondria and the main enzymes involved in the corticosterone, aldosterone and catecholamine synthesis. The results showed that CBG is synthesized in the adrenal gland. CBG-deficiency reduced the expression of ACTH receptor, SRB1 and the main genes involved in the adrenal hormones synthesis, stronger in females resulting in the loss of sexual dimorphism in corticosteroid adrenal synthesis, despite corticosterone content in adrenal glands from CBG-deficient females was similar to wildtype ones. In conclusion, these results point to an unexplored and relevant role of CBG in the adrenal gland functionality related to corticosterone production and release.

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. ACTH receptor is a subtype of the melanocortin receptor family which is a member of the G-protein coupled receptor (GPCR) superfamily. ACTH receptor has unique characteristics among MCRs. α-MSH, β-MSH, γ-MSH and ACTH are agonists for MCRs but only ACTH is the agonist for ACTH receptor. In addition, the melanocortin receptor accessory protein (MRAP) is required for ACTH receptor expression at cell surface and function. In this review, we summarized the information available on the relationship between ACTH and ACTH receptor and provide the latest understanding of the molecular basis of the ACTH receptor responsible for ligand selectivity and function.

The melanocortin-2-receptor (MC2R), also known as the ACTH receptor, is a critical component of the hypothalamic–pituitary–adrenal axis. The importance of MC2R in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency (FGD), a potentially fatal disease characterised by isolated cortisol deficiency. MC2R mutations cause ~25% of cases. The discovery of a MC2R accessory protein MRAP, mutations of which account for ~20% of FGD, has provided insight into MC2R trafficking and signalling. MRAP is a single transmembrane domain accessory protein highly expressed in the adrenal gland and essential for MC2R expression and function. Mouse models helped elucidate the action of ACTH. The Mc2r-knockout (Mc2r − / − ) mice was the first mouse model developed to have adrenal insufficiency with deficiencies in glucocorticoid, mineralocorticoid and catecholamines. We recently reported the generation of the Mrap − / − mice which better mimics the human FGD phenotype with isolated glucocorticoid deficiency alone. The adrenal glands of adult Mrap − / − mice were grossly dysmorphic with a thickened capsule, deranged zonation and deranged WNT4/beta-catenin and sonic hedgehog (SHH) pathway signalling. Collectively, these mouse models of FGD highlight the importance of ACTH and MRAP in adrenal progenitor cell regulation, cortex maintenance and zonation.

Maternal stress can impair foetal development and program sex-specific disease outcomes in offspring through the actions of maternally produced glucocorticoids, predominantly corticosterone (Cort) in rodents. We have demonstrated in mice that male but not female offspring prenatally exposed to Cort (33 µg/kg/h for 60 h beginning at E12.5) develop cardiovascular/renal dysfunction at 12 months. At 6 months of age, renal function was normal but male offspring had increased plasma aldosterone concentrations, suggesting that altered adrenal function may precede disease. This study investigated the long-term impact of prenatal exposure to Cort on adrenal growth, morphology and steroidogenic capacity as well as plasma Cort concentrations in offspring at postnatal day 30 (PN30), 6 months and 12 months of age. Prenatal Cort exposure decreased adrenal volume, particularly of the zona fasciculata, in male offspring at PN30 but increased both relative and absolute adrenal weight at 6 months of age. By 12 months of age, male Cort-exposed offspring had reduced absolute adrenal weight in association with increased adrenal plaque deposition (lipogenic pigmentation). Plasma Cort concentrations were elevated in male 6-month offspring but not at other ages. mRNA expression of Mc2r (ACTH receptor) was increased in males at PN30, and Cyp11a1 expression was decreased at 6 and 12 months of age. There were no changes in the adrenals of female Cort-exposed offspring. This study demonstrates that prenatal Cort exposure induces offspring adrenal gland dysfunction in an age- and sex-specific manner, which may contribute to long-term programmed disease in male offspring after maternal stress.

Background and purpose α- and β-melanocyte-stimulating hormones (MSH) are derived from pro-opiomelanocortin (POMC) and are the natural agonist ligands of the melanocortin 4 receptor, a key regulator of energy homeostasis. Recent rodent and human data have implicated the MAGEL2 gene, which may regulate activation of POMC neurons, as a significant contributor to the metabolic symptoms observed in Prader-Willi Syndrome (PWS). Firstly, patients with protein truncating mutations in MAGEL2 exhibit numerous clinical characteristics of PWS. Secondly, Magel2-null mice may not normally activate MC4 receptors, as they are defective in the activation of their POMC neurons and hence may fail to normally release the POMC-derived MC4 receptor agonist ligands α- and β-MSH. Magel2-null mice represent a tractable animal model for the metabolic and appetitive imbalance seen in patients with PWS. Experimental approach We tested a dose titration of the MC4 receptor agonist setmelanotide, in development for rare monogenic forms of obesity, in Magel2-null mice. Key results We show that Magel2-null mice are hypersensitive to the appetite suppressing and metabolic effects of setmelanotide. Conclusion and implications Setmelanotide may be a useful investigational hormone/neuropeptide replacement therapy for PWS and rare monogenic forms of obesity exhibiting impaired function of POMC neurons.

Within any population, the cortisol response to ACTH covers a considerable range. High responders (HRs) exhibit a greater cortisol secretory response to stress or ACTH, compared with individuals classified as low cortisol responders (LRs). We administered ACTH (0.2 μg/kg, iv) to 160 female sheep and selected subpopulations of animals as LR and HR. In the present study, we aimed to characterize the hypothalamo-pituitary-adrenal axis in HR and LR and to identify factors that underlie the differing cortisol responses to ACTH. Hypothalami, pituitaries, and adrenals were collected from nonstressed HR and LR ewes. Expression of genes for CRH, arginine vasopressin (AVP), oxytocin, glucocorticoid receptor, and mineralocorticoid receptor were measured by in situ hybridization in the paraventricular nucleus of the hypothalamus, and proopiomelanocortin (POMC) gene expression was measured in the anterior pituitary. Expression of CRH, AVP, and POMC was higher in HR, with no differences in either glucocorticoid receptor or mineralocorticoid receptor expression. Oxytocin expression was greater in LR. In the adrenal gland, real-time PCR analysis indicated that expression of the ACTH receptor and a range of steroidogenic enzymes was similar in HR and LR. Adrenal weights, the cortex to medulla ratio and adrenal cortisol content were also similar in LR and HR. In conclusion, LR and HR display innate differences in the steady-state expression of CRH, AVP, oxytocin, and POMC, indicating that selection for cortisol responsiveness identifies distinct subpopulations that exhibit innate differences in the gene expression/function of hypothalamo-pituitary-adrenal axis markers.

AimsRetinoic acid has recently yielded promising results in the treatment of Cushing's disease, i.e., excess cortisol secretion due to a pituitary corticotropin (ACTH)-secreting adenoma. In addition to its effect on the tumoral corticotrope cell, clinical results suggest an additional adrenal site of action. Aim of this study was to evaluate whether retinoic acid modulates cortisol synthesis and secretion by human adrenals in vitro.Main methodsPrimary cultures from 10 human adrenals specimens were incubated with 10nM, 100nM and 1μM retinoic acid with and without 10nM ACTH for 24h. Cortisol levels were measured by radioimmunoassay and CYP11A1, STAR and MC2R gene expression analyzed by real-time PCR.Key findingsRetinoic acid increased cortisol secretion (149.5±33.01%, 151.3±49.45% and 129.3±8.32% control secretion for 10nM, 100nM and 1μM respectively, p

Metformin is recommended as one of the first-line drugs for the treatment of type 2 diabetes and the metabolic syndrome. In addition to its insulin sensitizing effects, it has been shown to attenuate androgen excess in women with polycystic ovary syndrome (PCOS) or congenital adrenal hyperplasia (CAH), as well as to ameliorate obesity. The mechanisms of metformin action seem manifold. Preclinical studies suggest that it inhibits the cellular stress response at the level of the mitochondrial OXPHOS system and through AMPK dependent and independent mechanisms. Recent studies have shown that metformin decreases ACTH secretion from pituitary and reduces ACTH-stimulated adrenal secretion. In this study we investigated its specific effect through the melanocortin receptor 2 (MC2R) on signaling targeting adrenal steroidogenesis. To assess this effect, we used mouse adrenal OS3 cells, which do not express the MC2R. Cells were transfected with the MC2R and stimulated by ACTH. Downstream cyclic AMP production was then assessed by a co-transfected cAMP-responsive vector producing luciferase that was measured by a dual luciferase assay. The amount of luciferase produced in this assay corresponds to the amount of receptor activation with varying amount of ACTH. The effect of metformin was then tested in this system. We found a significant inhibition of ACTH induced MC2R activation and signaling with 10 mM metformin. The ACTH concentration response curve (CRC) was half-log shifted and a ∼30 % reduction in maximum receptor response (Rmax) to ACTH in presence of metformin was observed. This effect was dose dependent with an IC50 of 4.2 mM. qRT-PCR analyses showed that metformin decreased ACTH induced MC2R expression. Metformin did not affect cell viability and basal cAMP levels. We also tested the effect of metformin on homologous melanocortin receptors (MCRs). No significant effect was found on MC1R and MC4R activity. However, a log shift of EC50 of ACTH stimulation on MC3R was observed with metformin treatment. Metformin also inhibited melanocortin stimulating hormone (αMSH) induced MC3R activity. In conclusion, we show that metformin acts on MC2R and MC3R signaling directly. The role of MC2R for steroidogenesis is well established. MC3R is involved in energy balance and seems to act as a rheostat when the metabolism is challenged. Our study may explain how metformin helps in weight loss and attenuates the excess response to ACTH in androgen excess disorders such as PCOS and CAH.

7 p., The pentapeptides methionine-enkephalin and leucine-enkephalin belong to the opioid family of peptides, and the non-opiate peptide adrenocorticotropin hormone (ACTH) to the melanocortin peptide family. Enkephalins/ACTH are derived from pro-enkephalin, pro-dynorphin or pro-opiomelanocortin precursors and, via opioid and melanocortin receptors, are responsible for many biological activities. Enkephalins exhibit the highest affinity for the δ receptor, followed by the μ and κ receptors, whereas ACTH binds to the five subtypes of melanocortin receptor, and is the only member of the melanocortin family of peptides that binds to the melanocortin-receptor 2 (ACTH receptor). Enkephalins/ACTH and their receptors exhibit a widespread anatomical distribution. Enkephalins are involved in analgesia, angiogenesis, blood pressure, embryonic development, emotional behavior, feeding, hypoxia, limbic system modulation, neuroprotection, peristalsis, and wound repair; as well as in hepatoprotective, motor, neuroendocrine and respiratory mechanisms. ACTH plays a role in acetylcholine release, aggressive behavior, blood pressure, bone maintenance, hyperalgesia, feeding, fever, grooming, learning, lipolysis, memory, nerve injury repair, neuroprotection, sexual behavior, sleep, social behavior, tissue growth and stimulates the synthesis and secretion of glucocorticoids. Enkephalins/ACTH are also involved in many pathologies. Enkephalins are implicated in alcoholism, cancer, colitis, depression, heart failure, Huntington's disease, influenza A virus infection, ischemia, multiple sclerosis, and stress. ACTH plays a role in Addison's disease, alcoholism, cancer, Cushing's disease, dermatitis, encephalitis, epilepsy, Graves' disease, Guillain-Barré syndrome, multiple sclerosis, podocytopathies, and stress. In this review, we provide an updated description of the enkephalinergic and ACTH systems.

Introduction A stressful environment may contribute to poor outcomes after TBI. The current study evaluates the impact of acute stress in a polytrauma rat model. Methods Rats were stressed by a 45-minute immobilization period before instrumentation under ketamine (t1). Polytrauma was produced by blast overpressure and controlled hemorrhage (t2). Rats were euthanized immediately after a 3 h simulated Medevac-transport time (t3) or after 72 h post-trauma (t4). Corticosterone, ACTH, and ACTH receptor gene expression were measured at these time points. Physiological parameters were monitored throughout the study. Results HR was higher in stressed compared to unstressed animals at t1. Corticosterone and ACTH levels were similar for all conditions at t1 and t2; ACTH and corticosterone became elevated in all groups at t3 and at t4, respectively. The ACTH receptor gene expression trended towards higher values at t4 for the stressed animals whether being injured or not. Survival after injury was 83% in both unstressed and stressed animals. Conclusion Overall, corticosterone was not significantly affected following acute stress in ketamine-anesthetized rats. Early mortality was primarily due to polytrauma and change in the animal’s biochemical parameters appeared at t4 post trauma. The findings indicate that ketamine-anesthesia and/or surgery may have overshadowed the effect of the initial stress.

The adrenal has been neglected in endocrine disruption regulatory testing strategy. The adrenal is a vital organ, adrenocortical insufficiency is recognised in life threatening "adrenal crises" and Addison's disease, and the consequences of off-target toxicological inhibition of adrenocortical steroidogenesis is well recognised in clinical medicine, where drugs such as aminoglutethimide and etomidate killed patients via unrecognised inhibition of adrenocortical steroidogenic enzymes (e.g. CYP11B1) along the cortisol and aldosterone pathways. The consequences of adrenocortical dysfunction during early development are also recognised in the congenital salt wasting and adrenogenital syndromes presenting neonatally, yet despite a remit to focus on developmental and reproductive toxicity mechanisms of endocrine disruption by many regulatory agencies (USEPA EDSTAC; REACH) the assessment of adrenocortical function has largely been ignored. Further, every step in the adrenocortical steroidogenic pathway (ACTH receptor, StAR, CYP's 11A1, 17, 21, 11B1, 11B2, and 3-hydroxysteroid dehydrogenase Δ4,5 isomerase) is known to be a potential target with multiple examples of chemicals inhibiting these targets. Many of these chemicals have been detected in human and wildlife tissues. This raises the question of whether exposure to low level environmental chemicals may be affecting adrenocortical function. This review examines the omission of adrenocortical testing in the current regulatory frameworks; the characteristics that make the adrenal cortex particularly vulnerable to toxic insult; chemicals and their toxicological targets within the adrenocortical steroidogenic pathways; the typical manifestations of adrenocortical toxicity (e.g. human iatrogenically induced pharmacotoxicological adrenal insufficiency, manifestations in typical mammalian regulatory general toxicology studies, manifestations in wildlife) and models of adrenocortical functional assessment. The utility of the in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled 'Endocrine disruptors and steroids'.

The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic–pituitary–adrenal axis.

The rapid release of cortisol from the adrenal cortex upon ACTH receptor activation plays an integral role in the stress response. It has been suggested that the quantitative control over adrenal steroidogenesis (quantity of total steroids produced) depends on the activities of cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein that supplies pregnenolone precursor to the pathway. The qualitative control (which steroids) then depends on the downstream steroidogenic enzymes, including cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17). In this review we focus on the relative contribution of P450c17 in the qualitative control of cortisol production with data collected from studies on South African Angora and Boer goats, as well as Merino sheep. Unique P450c17 genotypes were identified in these breeds with isoforms differing only with a couple of single amino acid residue substitutions. This review demonstrates how molecular and cellular differences relating to P450c17 activity can affect physiological and behavioural responses.

Following the cloning of the melanocortin receptor and agouti protein genes, a model was developed for the central melanocortin system with respect to the regulation of energy and glucose homeostasis. This model comprised leptin regulation of melanocortin peptides and agouti-related peptide (AgRP) produced from central pro-opiomelanocortin (POMC) and AgRP neurones, respectively, as well as AgRP competitive antagonism of melanocortin peptides activating melanocortin 4 receptor (MC4R) to Gαs and the cAMP signalling pathway. In the last decade, there have been paradigm shifts in our understanding of the central melanocortin system as a result of the application of advanced new technologies, including Cre-LoxP transgenic mouse technology, pharmacogenetics and optogenetics. During this period, our understanding of G protein coupled receptor signal transduction has also dramatically changed, such that these receptors are now known to exist in the plasma membrane oscillating between various inactive and active conformational states, and the active states signal through G protein-dependent and G protein-independent pathways. The present review focuses on evidence obtained over the past decade that has changed our understanding of POMC gene expression and regulation in the central nervous system, POMC and AgRP neuronal circuitry, neuroanatomical functions of melanocortin receptors, melanocortin 3 receptor (MC3R) and MC4R, and signal transduction through MC3R and MC4R.

Objectives: To characterize the dynamics of the pituitary-adrenal interaction during the course of coronary artery bypass grafting both on and off pump. Since our data pointed to a major change in adrenal responsiveness to adrenocorticotropic hormone, we used a reverse translation approach to investigate the molecular mech-anisms underlying this change in a rat model of critical illness.Design: Clinical studies: Prospective observational study. Animal studies: Controlled experimental study.Setting: Clinical studies: Cardiac surgery operating rooms and critical care units. Animal studies: University research laboratory.Subjects: Clinical studies: Twenty, male patients. Animal studies: Adult, male Sprague-Dawley rats.Interventions: Clinical studies: Coronary artery bypass graft—both on and off pump. Animal studies: Injection of either lipopolysac-charide or saline (controls) via a jugular vein cannula.Measurements and Main Results: Clinical studies: Blood sam-ples were taken for 24 hours from placement of the first venous access. Cortisol and adrenocorticotropic hormone were mea-sured every 10 and 60 minutes, respectively, and corticosteroid-binding globulin was measured at the beginning and end of the 24-hour period and at the end of operation. There was an initial rise in both levels of adrenocorticotropic hormone and cortisol to supranormal values at around the end of surgery. Adrenocor-ticotropic hormone levels then returned toward preoperative val-ues. Ultradian pulsatility of both adrenocorticotropic hormone and cortisol was maintained throughout the perioperative period in all individuals. The sensitivity of the adrenal gland to adrenocortico-tropic hormone increased markedly at around 8 hours after sur-gery maintaining very high levels of cortisol in the face of “basal” levels of adrenocorticotropic hormone. This sensitivity began to return toward preoperative values at the end of the 24-hour sam-pling period. Animal studies: Adult, male Sprague-Dawley rats were given either lipopolysaccharide or sterile saline via a jugular vein cannula. Hourly blood samples were subsequently collected for adrenocorticotropic hormone and corticosterone measure-ment. Rats were killed 6 hours after the injection, and the adrenal glands were collected for measurement of steroidogenic acute regulatory protein, steroidogenic factor 1, and dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 messenger RNAs and protein using real-time quantitative polymerase chain reaction and Western immunoblotting, respec-tively. Adrenal levels of the adrenocorticotropic hormone receptor (melanocortin type 2 receptor) messenger RNA and its accessory protein (melanocortin type 2 receptor accessory protein) were also measured by real-time quantitative polymerase chain reac-tion. In response to lipopolysaccharide, rats showed a pattern of

Mechanisms regulating differences in cortisol responsiveness between low (LR) and high response (HR) individuals have been poorly studied. In this context, we aimed to study key regulatory processes in cortisol dynamics at the head kidneys of LR and HR European sea bass. To do so, resting plasma cortisol and ACTH concentrations were quantified in these fish. Additionally, the head kidneys of these individuals were superfused through an in vitro superfusion system and stimulated with the same amount of ACTH to assess their cortisol biosynthetic capacity. Moreover, the expression of important genes in cortisol regulation was assessed. Results showed that LR fish had lower resting cortisol concentrations than HR, although no differences existed in the circulating levels of ACTH. Additionally, the biosynthetic capacity of HR was higher than that of LR fish when in vitro stimulated with ACTH. At the molecular level, a statistically significant 3.4-fold higher expression of the ACTH receptor, mc2r, and a 2.3-fold, though not significant, higher expression of 11β-hydroxylase (cyp11b1), an enzyme involved in cortisol biosynthesis, was observed in the HR fish. Finally, a statistically significant 1.3-fold lower expression of 11β-hydroxysteroid dehydrogenase 2 (hsd11b2), an enzyme involved in cortisol inactivation, was observed in HR when compared to LR fish. Therefore, it was for the first time indicated that cortisol dynamics can also be regulated at the post-production level in the head kidney. Collectively, our results highlight the crucial role of the interrenal tissue in the regulation of differences in cortisol response between LR and HR sea bass individuals.

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease caused by resistant of ACTH receptor at adrenal cortex leading to (usually) isolated glucocorticoid deficiency with normal mineralocorticoid secrete. Patients with FGD usually presented in neonatal–childhood period with signs/symptoms of glucocorticoid deficiency such as hypoglycemia, hyperpigmentation, Failure to thrive, shock and death if treatment was delayed. Labs usually revealed high ACTH, low cortisol but normal 17 OHP, electrolyte, androgen. Here we describe a 3-yearsold Saudi girl, with history of progressive hyperpigmentation for first year of life, but no history of hypoglycaemia or neonatal jaundice, no history of a lacrimation or dysphagia and positive similar family history. She had generalized Hyperpigmentation with normal female genitalia. Her cortisol was low with high ACTH level, but normal electrolyte 17, Hydroxyprogesterone, aldosterone, renin, androgen. Familial Glucocorticoid Deficiency was diagnosed, and maintenance dose of hydro cortisol was started, and patient pigmentation was improved few week latter.

ACTH is a major hormone of the stress axis or hypothalamic-pituitary-adrenal (HPA) axis. It is derived from pro-opiomelanocortin (POMC) the precursor to the melanocortin family of peptides. POMC produces the biologically active melanocortin peptides via a series of enzymatic steps in a tissue-specific manner, yielding the melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH) and ?-endorphin. The melanocortin system plays an imperative role in energy expenditure, insulin release and insulin sensitivity. Bone marrow derived mesenchymal stem cells circulate in the blood stream and as progenitor cells have the potential to differentiate into many cell types such as osteoblasts, chondrocytes and adipocytes. Here we examine the effects of ACTH on the mouse D1 bone-marrow derived MSC. ACTH significantly increased lipid accumulation during the adipogenic differentiation of D1 cells in a concentration- dependent manner. ACTH also shifts the temporal pattern of D1 adipogenic differentiation to the left i.e. differentiation occurs earlier with ACTH treatment. No significant differences in protein expression of peroxisome proliferator-activated receptor gamma (PPAR-?2), a regulating transcription factor of adipogenesis were found. Therefore the effects of ACTH are suggested to be mediated by an alternative pathway. Overall the results indicate a connection between increased adipose deposition and the elevated circulating ACTH associated with stress.

The effect of Roundup® on adrenal gland steroidogenesis and signaling pathway associated with steroid production was investigated. Doses of 10, 50, 100 and 250mg/kg bw/d Roundup® were administered for two weeks to adult male rats. The 10mg/kg bw/d dose which reduced circulatory corticosterone levels, but did not change food consumption and body weight, was selected for further study. The expression of cholesterol receptor (low density lipoprotein receptor), de novo cholesterol synthesis enzyme (3-hydroxy-3-methylglutaryl-coenzyme A synthase), hormone-sensitive lipase, steroidogenic acute regulatory protein (StAR) mRNA and phosphorylated form was decreased. Adrenocorticotropic hormone receptor (ACTH), melanocortin-2 receptor, expression was not changed but circulatory ACTH levels and adrenal cortex protein kinase A (PKA) activity were reduced. Surprisingly, exogenous ACTH treatment rescued steroidogenesis in Roundup®-treated animals. Apoptosis was evident at 250mg/kg bw/d, but not at 10mg/kg bw/d dose. These results suggest that Roundup® may be inhibitory to hypothalamic–pituitary axis leading to reduction in cyclic adenosine monophosphate (cAMP)/PKA pathway, StAR phosphorylation and corticosterone synthesis in the adrenal tissue.

Orexins peptides exert a prominent role in arousal-related processes including stress responding, by activating orexin-1 (OX1R) and orexin-2 (OX2R) receptors located widely throughout the brain. Stress or orexin administration stimulates hyperarousal, adrenocorticotropic hormone (ACTH) and corticosterone release, and selective OX1R blockade can attenuate several stress-induced behavioral and cardiovascular responses but not the hypothalamic-pituitary-adrenal (HPA) axis activation. As opposed to OX1R, OX2R are preferentially expressed in the paraventricular hypothalamic nucleus which is involved in the HPA axis regulation. In the present study, we investigated the effects of a psychological stress elicited by cage exchange on ACTH release in two murine models (genetic and pharmacological) of selective OX2R inhibition. Cage exchange-induced stress produced a significant increase in ACTH serum levels. Mice lacking the OX2R exhibited a blunted stress response. Stress-induced ACTH release was absent in mice pre-treated with the selective OX2R antagonist JNJ-42847922 (30 mg/kg po), whereas pre-treatment with the dual OX1/2R antagonist SB-649868 (30 mg/kg po) only partially attenuated the increase of ACTH. To assess whether the intrinsic and distinct sleep-promoting properties of each antagonist could account for the differential stress response, a separate group of mice implanted with electrodes for standard sleep recording were orally dosed with JNJ-42847922 or SB-649868 during the light phase. While both compounds reduced the latency to non-rapid eye movement (NREM) sleep without affecting its duration, a prevalent REM-sleep promoting effect was observed only in mice treated with the dual OX1/2R antagonist. These data indicate that in a psychological stress model, genetic or pharmacological inhibition of OX2R markedly attenuated stress-induced ACTH secretion, as a separately mediated effect from the NREM sleep induction of OX2R antagonism.

Chronic ACTH excess leads to chronic cortisol excess, without escape phenomenon, resulting in Cushing’s syndrome. Excess adrenal androgens also occur: in females, they will overcompensate the gonadotrophic loss, inducing high testosterone; in males, they will not compensate it, inducing low testosterone. Chronic ACTH excess leads to chronic adrenal mineralocorticoid excess and low aldosterone levels: after an acute rise, aldosterone plasma levels resume low values after a few days when ACTH is prolonged. Two other mineralocorticoids in man, cortisol and 11 deoxycorticosterone (DOC), at the zona fasciculata, will not escape the long-term effect of chronic ACTH excess and their secretion rates will remain elevated in parallel. Over all, the concomitant rise in cortisol and 11 DOC will more than compensate the loss of aldosterone, and eventually create a state of chronic mineralocorticoid excess, best evidenced by the accompanying suppression of the renin plasma levels, a further contribution to the suppression of aldosterone secretion. Prolonged in vivo stimulation with ACTH leads to an increase in total adrenal protein and RNA synthesis. Cell proliferation is indicated by an increase in total DNA the resulting adrenocortical hyperplasia participates in the amplified response of the chronically stimulated gland, and the weight of each gland can be greatly increased. The growth-stimulatory effect of ACTH in vivo most likely proceeds through the activation of a local and complex network of autocrine growth factors and their own receptors; a number of compounds, including non-ACTH proopiomelanocortin peptides such as γ3-MSH, have been shown to exert some adrenocortical growth effect.

Objectives:The cortisol response during critical illness varies widely among patients. Our objective was to examine single nucleotide polymorphisms in candidate genes regulating cortisol synthesis, metabolism, and activity to determine if genetic differences were associated with variability in the c

Studies have indicated significant pubertal-related differences in hormonal stress reactivity. We report here that prepubertal (30 days) male rats display a more protracted stress-induced corticosterone response than adults (70 days), despite showing relatively similar levels of adrenocorticotropic hormone (ACTH). Additionally, we show that adrenal expression of the ACTH receptor, melanocortin 2 receptor ( Mc2r ), is higher in prepubertal compared to adult animals, and that expression of melanocortin receptor accessory protein ( Mrap ), a molecule that chaperones MC2R to the cell surface, is greater in prepubertal males following stress. Given that these data suggest a pubertal shift in adrenal sensitivity to ACTH, we directly tested this possibility by injecting prepubertal and adult males with 6.25 or 9.375 μg/kg of exogenous rat ACTH and measured their hormone levels 30 and 60 min post-injection. As these doses resulted in different circulating levels of ACTH at these two ages, we performed regression analyses to assess the relationship between circulating ACTH and corticosterone concentrations. We found no difference between the ages in the correlation between ACTH and corticosterone levels at the 30 min time point. However, 60 min following the ACTH injection, we found prepubertal rats had significantly higher corticosterone concentrations at lower levels of ACTH compared to adults. These data suggest that prolonged exposure to ACTH leads to greater corticosterone responsiveness prior to puberty, and indicate that changes in adrenal sensitivity to ACTH may, in part, contribute to the protracted hormonal stress response in prepubertal rats.

Stimulation of the serotonin 1A (5-HT1A) receptor subtype by 5-HT has been shown to result in an elevation in plasma corticosteroid levels in both mammals and several species of teleost fish, including the Gulf toadfish (Opsanus beta); however, in the case of teleost fish, it is not clearly known at which level of the hypothalamic-pituitary-interrenal axis the 5-HT1A receptor is stimulated. Additionally, previous investigations have revealed that chronic elevations of plasma cortisol mediate changes in brain 5-HT1A receptor mRNA and protein levels via the glucocorticoid receptor (GR); thus, we hypothesized that the function of centrally activated 5-HT1A receptors is reduced or abolished as a result of chronically elevated plasma cortisol levels and that this response is GR mediated. Our results are the first to demonstrate that intravenous injection of the 5-HT1A receptor agonist, 8-OH-DPAT, stimulates a significant increase in corticotropin-releasing factor (CRF) precursor mRNA expression in the hypothalamic region and the release of adrenocorticotropic hormone (ACTH) from the pituitary of teleost fish compared to saline-injected controls. We also provide evidence that cortisol, acting via GRs, attenuates the 5-HT1A receptor-mediated secretion of both CRF and ACTH.

Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

The melanocortin system integrates different agonists, competitive or inverse agonists, and receptors. Recent investigations have also discovered a specific system of melanocortin receptor accessory proteins (MRAPs) that are involved in the regulation of the functional expression of these receptors. MRAP1 mutations are responsible for type 2 familial glucocorticoid deficiency (FGD2), a rare autosomal disorder characterized by high plasma adrenocorticotropin hormone (ACTH) levels but severe cortisol deficiency. ACTH binds melanocortin 2 receptor (MC2R), a G protein-coupled receptor, in the adrenal gland to promote corticosteroid synthesis. In the absence of MRAP1, MC2R cannot translocate from the endoplasmic reticulum to the plasma membrane and ACTH-induced signaling is extinguished. A second MRAP protein, called MRAP2, also modulates MC2R activity. MRAPs also interact with the other melanocortin receptors, adjusting their pharmacological properties. In this paper, we briefly review the MRAP system and its interaction with melanocortin receptors. © 2013 Elsevier Inc., Grants AGL2010-22247-C03-01 and CSD 2007-00002 from Spanish Science and Education Ministry (MEC) to JMC-R. Additional funding was obtained from the >Generalitat Valenciana> (research grant PROMETEO 2010/006). MJA and RC are recipient of a >Juan de la Cierva> research contract and FPI fellowship, respectively, from MINECO.

Cushing disease (CD) is a life-threatening disorder attributed to excess pituitary tumor-derived adrenocorticotrophic hormone (ACTH) and adrenal steroid secretion caused by pituitary tumors. Whereas CD was first described in 1932, the underlying genetic basis driving tumor growth and ACTH secretion remains unsolved. Here, we show that testicular orphan nuclear receptor 4 (TR4, nuclear receptor subfamily 2, group C, member 2) is overexpressed in human corticotroph tumors as well as in human and mouse corticotroph tumor cell lines. Forced overexpression of TR4 in both human and murine tumor cells increased proopiomelanocortin transcription, ACTH secretion, cellular proliferation, and tumor invasion rates in vitro. Conversely, knockdown of TR4 expression reversed all phenotypes. Mechanistically, we show that TR4 transcriptionally activates proopiomelanocortin through binding of a direct repeat 1 response element in the promoter, and that this is enhanced by MAPK-mediated TR4 phosphorylation. In vivo, TR4 overexpression promotes murine corticotroph tumor growth as well as enhances ACTH and corticosterone production, whereas TR4 knockdown decreases circulating ACTH and corticosterone levels in mice harboring ACTH-secreting tumors. Our findings directly link TR4 to the etiology of corticotroph tumors, hormone secretion, and cell growth as well as identify it as a potential target in the treatment of CD.

The Adrenocorticotropic hormone (ACTH) and Pro-opimelanocortin (POMC) 1-28N-terminal peptide (N-POMC(1-28)) have been shown to act as an adrenal mitogen in vivo. A possible role for cyclin E in the zona glomerulosa (ZG) proliferation, following ACTH and/or N-POMC(1-28) administration, has been previously demonstrated. In this study, we investigated the effect of ACTH and N-POMC(1-28) on the expression of adrenal cortex proteins related to cell cycle control such as cyclins D and P27(kip1). The administration of N-POMC upregulated cyclin D1 and D2 expression in the outer zone of the adrenal cortex; cyclin D3 expression was upregulated in the cortex inner zone even after administration of ACTH. Both ACTH and N-POMC peptides induced a decrease in the P27(kip1) expression in the ZG. These novel findings suggest that the POMC-derivate peptides, ACTH and N-POMC, promote proliferation in the adrenal cortex by upregulating the D2 and D3 cyclins and downregulating the P27(kip1) expression.

The adrenocorticotropic hormone ACTH is a pituitary hormone derived from a larger peptide, the proopiomelanocortine (POMC), as are the MSHs (α-MSH, β-MSH and γ-MSH) and the β-LPH-related polypeptides (Figure 1A). ACTH drives adrenal steroidogenesis and growth of the adrenal gland. ACTH is a 39 amino acids polypeptide that binds and activates its cognate receptor (melanocortin receptor 2, MC2R) through the two regions H6F7R8W9 and K15K16R17R18P19. Most POMC-derived polypeptides contain the H6F7R8W9 sequence that is conserved through evolution. This explains the difficulties in developing selective agonists or antagonists to the melanocortin receptors. In this review, we will discuss the clinical aspects of the role of ACTH in physiology and disease, and potential clinical use of selective ACTH antagonists.

Glucocorticoid (GC) production is physiologically regulated through a negative feedback loop mediated by the GC, which appears disrupted in several pathological conditions. The inability to perform negative feedback of the hypothalamus-pituitary-adrenal (HPA) axis in several diseases is associated with an overproduction of reactive oxygen species (ROS); however, nothing is known about the effects of ROS on the functionality of the HPA axis during homeostasis. This study analyzed the putative impact of antioxidants on the HPA axis activity and GC-mediated negative feedback upon the HPA cascade. Male Wistar rats were orally treated with N-acetylcysteine (NAC) or vitamin E for 18 consecutive days. NAC-treated rats were then subjected to a daily treatment with dexamethasone, which covered the last 5 days of the antioxidant therapy. We found that NAC and vitamin E induced an increase in plasma corticosterone levels. NAC intensified MC2R and StAR expressions in the adrenal and reduced GR and MR expressions in the pituitary. NAC also prevented the dexamethasone-induced reduction in plasma corticosterone levels. Furthermore, NAC decreased HO-1 and Nrf2 expression in the pituitary. These findings show that antioxidants induce hyperactivity of the HPA axis via upregulation of MC2R expression in the adrenal and downregulation of GR and MR in the pituitary.

Adrenocorticotropic hormone (ACTH) is synthesized within the corticotroph cells of the anterior pituitary lobe from processing of proopiomelanocortin (POMC), the precursor molecule. New assays have solved many of the problems in the measurement of ACTH. The secretion of ACTH is controlled by an inherent diurnal rhythmicity; it is augmented by noxious stimuli that are neurally, hormonally, and biochemically mediated, which is termed stress (open-loop component), and is inhibited by glucocorticoids (closed-loop, negative feedback). The adrenal cortex is the principal target organ for ACTH where it stimulates the synthesis and release of steroids from adrenocortical cells.

Adrenocorticotrophin (ACTH) is the anterior pituitary mediator of the hypothalamic–pituitary–adrenal axis that regulates responses to a variety of stressors, including hypoglycemia, psychological stressors such as fear, and physical stressors such as hypovolemia. ACTH is the 39-amino-acid product of proopiomelanocortin (POMC), which is principally synthesized in anterior pituitary corticotrophs, neurons of the mediobasal hypothalamus, and skin melanocytes. In each of these cell types, POMC is processed to several melanocortins: ACTH (in the pituitary), α-MSH (in the skin and hypothalamus), and β-endorphin (in all three). ACTH, in response to hypothalamic corticotrophin-releasing hormone, is secreted and binds to the adrenal melanocortin 2 receptor (MC2R), a G protein-coupled receptor that signals through cyclic AMP to stimulate cortisol production and secretion. The main action of cortisol is to maintain adequate body fuel supplies and blood pressure during times of stress. In response to ultraviolet light exposure, skin α-MSH is released and binds to MC1R, which stimulates production of melanin to increase skin pigmentation. α-MSH in the hypothalamus, in response to leptin, is released from neurons to act through MC4R receptors in hypothalamic paraventricular nuclei to decrease appetite and increase energy expenditure. Diseases involving ACTH are due to pituitary or ectopic tumors that secrete excessive ACTH, faulty transcription of the POMC gene, abnormal cleavage of the POMC precursor, or defects in signaling of POMC products via (at least) MC1R, MC2R, or MC4R. MC2R and MC4R require an accessory protein for their functions, melanocortin receptor 2 accessory protein (MRAP), and MRAP2, respectively, and mutations in these proteins impair associated receptor function.

When considering the interactions between the melanocortin peptides (i.e., ACTH, α-MSH, β-MSH, γ-MSH) and the melanocortin receptors (i.e., MC1R, MC2R, MC3R, MC4R, MC5R), it appears that the structure/function relationship between ACTH and MC2R is the most complicated. Human ACTH(1-24) and the human melanocortin-2 receptor provide a useful model system for understanding how ACTH emerged as the sole ligand for the melanocortin-2 receptor of bony vertebrates. This review will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for activation of the melanocortin-2 receptor. In addition, observations on the unique activation features of the melanocortin-2 receptor, as revealed from studies on Familial Glucocorticoid Deficiency, will be considered. Finally, the evolutionary implications of the relationship between MC2R and MRAP1 will be discussed.

Acrolein is a small unsaturated aldehyde and can be found in a wide range of resources including all types of smoke and exhaust gases from gasoline engines. Although the toxicity and damage of acrolein have been recognized, the action mechanisms of acrolein, especially that of acrolein on the response of stresshormones are still unclear. The present study hypothesized that administration of acrolein altered the secretion of both adrenocorticotropin (ACTH) and corticosterone via the regulation of steroid biosynthetic pathway in rat zona fasciculata-reticularis (ZFR) cells. Both in vivo and in vitro approaches were uased. In the in vivo study, intraperitonal injection of acrolein (2 mg/ml/kg) once daily for 1 or 3 days resulted in a reduction of plasma levels of ACTH and corticosterone as well as the intracellular cAMP and ACTH-induced secretion of corticosterone. The protein expression of ACTH receptor (ACTHR) in rat ZFR cells was also reduced by 40-60% after treatment of acrolein for 1 day and 3 days, respectively. In the in vitro study, rat ZFR cells were prepared and chanllenged with ACTH (10 -9 M), forskolin (an adenylyl cyclase activitior, 10 -5 M), 8-Br-cAMP (a permeable synthetic cAMP, 5x10 -5 M), 25-OH-cholesterol (10 -5 M) ± trilostane (an inhibitor of 3β-hydroxysteroid dehydrogenase, 3β-HSD, 10 -5 M). The evoked release of corticosterone by ACTH, forskolin, 8-Br-cAMP and the induced release of pregnenolone in response to 25-OH-cholesterol plus triolostane were decreased. Since the accumulation of pregnenolone after blocking 3β-HSD by trilostane represents the activity of P450 scc, therate-limiting step of steroid biosynthesis, we suggest that not only the cAMP pathway was inhibited, but also the enzyme activity of P450 scc was attenuated following administration of acrolein. Although insignificant, the protein expression of steroidogenic acute regulatory protein (StAR) was decreased by 40% in ZFR cells after treatment of acrolein in vivo . Incubation of ZFR cells with acrolein (10 -9 ~10 -7 M) also decreased the in vitro release of corticosterone. These results suggest that administration of acrolein inhibited corticosterone production via the attenuation of cAMP pathway, StAR protein expression, and the enzyme activity of P450 scc. The attenuation of protein expression of ACTHR (also named melanocortin 2 receptor, MC2R) and reduced secrection of ACTH indicated that the hypothalamus-pituitary-adrenal (H-P-A) axis was also down- regulated by the administration of acrolein.

Adrenocorticotropic hormone (ACTH) is the only medicine for treating infantile spasms, however, it is catabolized rapidly. In order to make an ACTH derivative with prolonged effects, we prepared genetically engineered wild type (WT) and mutant ACTH candidates based on protease database analysis, and compared their stability and pharmacological effects. For analysis of stability, serum concentration of WT and mutant ACTH candidates were tested at different time after intravenous injection, and elimination curves were calculated to compare pharmacokinetic properties of WT and E5D-mutant ACTH. For comparison of their pharmacological effects, levels of glucocorticoids (GC) in the blood serum and secreted from cultured Y1 mouse adrenal cells were tested, and their effects on the signaling pathway mediating the expression of genes critical for GC synthesis were analyzed. The effects of ACTHs on transcription levels of the genes involved in GC synthesis were tested by qPCR. The blood concentration of E5D ACTH is higher than the WT after injection, and E5D mutation increased the t1/2 and AUC of ACTH. Pharmacological experiments showed that the effects of E5D and Y2S mutant ACTH on the production of GC and the critical signal transduction were equivalent to those of WT. WT, E5D and Y2S ACTH also have similar effects on the transcriptional levels of the genes for GC synthesis, including STAR, P450-scc, 3β-HSD, and SF-1. The stability of E5D mutant ACTH is higher than WT ACTH. The pharmacological effects of E5D ACTH is equivalent to those of WT ACTH.

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.

Hypothalamus-pituitary-adrenal (HPA) axis, as the key moderator in energy metabolism, plays an important role in diabetes. The endogenous cannabinoid system (eCBs) involves in neuronal functions, and simultaneously cannabinoid receptors are almost expressed in all regions of the hypothalamus according to a spate of reports. However, few data investigate the changes of eCBs and HPA axis in type 2 diabetes. In this study, five diabetes mellitus rhesus monkeys, five prediabetes rhesus monkeys and five healthy rhesus monkeys were observed. In the present study, we detected cell swelling and necrosis extensively in the paraventricular nucleus (PVN) and neurohypophysis in prediabetes and overt diabetes monkeys. The adrenocorticotropic hormone in the pituitary gland, adrenocorticotropic hormone receptor, and 11β-hydroxysteroid dehydrogenase in the adrenal gland were all hyper-secreted and expressed from healthy to overt diabetes. Meanwhile, the cortisol concentration in the adrenal gland was increased along with the progress of diabetes. It could be concluded that hyperfunction of the HPA axis exists in the type 2 Diabetes pathogenesis. However, we also found a weakened expression and secretion of corticotrophin releasing hormone and glucocorticoids receptor in PVN. The expression of corticotropin releasing hormone receptor 1 in pituitary gland decreased in prediabetes monkeys, but increased in overt diabetes monkeys. The downregulation of cannabinoid receptor 1 and upregulation of monoglycerol lipase and fatty acid amide hydrolase in PVN was involved in the pathogenesis of type 2 diabetes. Collectively, we can conclude that changes in endocannabinoid hydrolase and cannabinoid receptor might indicate the effect of downregulation of eCBs. It can be assumed that hyper-function of the HPA axis from healthy to overt diabetes is due to the undermining inhibition of eCBs. However, the regulatory mechanism of eCBs targets on the HPA axis need to be further explored.

Objective To test the hypothesis that local activation of melanocortin receptor(s) by adrenocorticotropic hormone (ACTH) could be responsible, at least in part, for its efficacy in human gouty arthritis. Methods Monosodium urate monohydrate (MSU) crystals were administered into rat knee joints either alone or with ACTH or a selective melanocortin type 3 receptor (MC3-R) agonist. Neutrophil migration, arthritis score, increases in joint size, and cytokine levels were measured over time. MC3-R expression on rat knee joint macrophages was monitored by electron microscopy and intracellular accumulation of cyclic adenosine monophosphate. Results MSU crystals produced a knee joint inflammation that was time dependent and was characterized by cell influx and cytokine release that was sensitive to treatment with classic anti-arthritic drugs (indomethacin, colchicine, dexamethasone). Local, but not systemic, ACTH had an antiinflammatory effect in normal rats, a dose that did not alter circulating corticosterone (5 μg). This treatment was also effective in adrenalectomized rats. Rat knee joint macrophages expressed functional MC3-R. The MC3-R antagonist (SHU9119, 10 μg) blocked ACTH antiinflammatory actions, whereas antiinflammatory activity was retained with a selective MC3-R agonist (γ2-melanocyte–stimulating hormone). Conclusion This research provides evidence for a separate mechanism of action of ACTH in experimental gouty arthritis and points to a novel antiinflammatory target (selective agonists at MC3-R) for clinical management of human gouty arthritis and possibly other chronic inflammatory conditions.