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Although it is known that stresses on females damage oocytes with increased production of stress hormones, whether corticotrophin-releasing hormone (CRH) or adrenocorticotropic hormone (ACTH) harm oocytes directly are largely unknown. We demonstrated that CRH exposure during in vitro maturation impaired competence of both pig and mouse cumulus-oocyte-complexes (COCs), and it impaired competence and induced apoptosis in pig cumulus-denuded oocytes (DOs) but not in mouse DOs. CRH receptor 1 was expressed in pig DOs and in cumulus cells (CCs) of both species but not in mouse DOs. In the presence of CRH, whereas mouse CCs underwent apoptosis, pig CCs did not. While pig CCs did, mouse CCs did not express CRH-binding protein. ACTH did not affect competence of either pig or mouse COCs or DOs although they all expressed ACTH receptor. Both pig and mouse CCs expressed steroidogenic acute regulatory protein (StAR), and ACTH enhanced their progesterone production while alleviating their apoptosis. Neither pig nor mouse DOs expressed StAR, but ACTH inhibited maturation-promoting factor and decelerated meiotic progression of DOs suggesting activation of protein kinase A (PKA). In conclusion, CRH impaired pig and mouse oocyte competence by interacting with CRH receptor and inducing CCs apoptosis, respectively. ACTH activated PKA in both DOs and CCs although it showed no effect on oocyte competence.

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.

Leydig cells (LC) are the main testicular androgen-producing cells. In eutherian mammals, two types of LCs emerge successively during testicular development, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Both display significant differences in androgen production and regulation. Using bulk RNA sequencing, we compared the transcriptomes of both LC populations to characterise their specific transcriptional and functional features. Despite similar transcriptomic profiles, a quarter of the genes show significant variations in expression between FLCs and ALCs. Non-transcriptional events, such as alternative splicing was also observed, including a high rate of intron retention in FLCs compared to ALCs. The use of single-cell RNA sequencing data also allowed the identification of nine FLC-specific genes and 50 ALC-specific genes. Expression of the corticotropin-releasing hormone 1 (Crhr1) receptor and the ACTH receptor melanocortin type 2 receptor (Mc2r) specifically in FLCs suggests a dual regulation of steroidogenesis. The androstenedione synthesis by FLCs is stimulated by luteinizing hormone (LH), CRH and ACTH whereas the testosterone synthesis by ALCs is dependent exclusively on LH. Overall, our study provides a useful database to explore LC development and function.

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.

The adrenocorticotropic hormone (ACTH) receptor, known as the melanocortin-2 receptor (MC2R), plays a key role in regulating adrenocortical function. MC2R is a subtype of the melanocortin receptor family and ACTH is only agonist for MC2R. Our previous result indicates that ACTH1-17 is the minimal peptide required for MC2R activation but DPhe7-ACTH1-17 has no activity at MC2R. In this study, we examined the molecular basis of the MC2R responsible for ligand selectivity using ACTH analogues and MC2R mutagenesis. Our results indicate that substitution of the 3TM of the MC2R with the corresponding region of the MC3R switches DPhe-ACTH1-17 from no activity to agonist. Further experiment indicates that substitution of the amino acid residue leucine to isoleucine in 112 (L112I) of the 3TM of the MC2R changes both DPhe-ACTH1-17 and ACTH1-15 from no activity to agonists. Surprisingly, mutation L112I switches α-MSH from no activity to agonist, suggesting that this residue plays a key role at MC2R for ligand ACTH or α-MSH selectivity.

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

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 the effect of metformin through its specific 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 human melanocortin receptor 2 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 indicating antagonism. This effect was dose dependent with an IC50 of 4.2 mM. Metformin did not affect cell viability and basal cAMP level under used conditions. We also tested the effect of metformin on homologous receptors (MCRs). No significant effect was found on MC1R and MC4R activity. However, a 2-log shift in ACTH EC50 was observed with MC3R. In conclusion, metformin seems to act 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 attenuates the excess response to ACTH and helps in weight loss and improving androgen excess in PCOS and CAH.

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.

G-protein coupled receptors (GPCRs) are regulated by numerous proteins including kinases, G-proteins, β-arrestins and accessory proteins. Several families of GPCR accessory proteins like Receptor Activity Modifying Proteins, Receptor Transporting Proteins and Melanocortin Receptor Accessory Proteins (MRAPs) have been identified as regulator of receptor trafficking, signaling and ligand specificity. The MRAP family contains two members, MRAP1 and MRAP2, responsible for the formation of a functional ACTH receptor and for the regulation of energy homeostasis respectively. Like all known GPCR accessory proteins, MRAPs are single transmembrane proteins, however, they form a unique structure since they assemble as an anti-parallel homodimer. Moreover, the accepted idea that MRAPs are specific regulators of melanocortin receptors was recently challenged by the discovery that MRAP2 inhibits the activity of prokineticin receptors. Recent studies are starting to explain the role of the unusual structure of MRAPs and to illustrate the importance of MRAP2 for the maintenance of both energy and glucose homeostasis. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

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.

It is well-established that infectious stress activates the hypothalamus–pituitary–adrenal axis leading to the production of pituitary adrenocorticotropin (ACTH) and adrenal glucocorticoids (GCs). Usually, GC synthesis is mediated by protein kinase A (PKA) signaling pathway triggered by ACTH. We previously demonstrated that acute murine Chagas disease courses with a marked increase of GC, with some data suggesting that GC synthesis may be ACTH-dissociated in the late phase of this parasitic infection. Alternative pathways of GC synthesis have been reported in sepsis or mental diseases, in which interleukin (IL)-1β, prostaglandin E2 (PGE2), and/or cAMP-activated guanine nucleotide exchange factor 2 (EPAC2) are likely to play a role in this regard. Accordingly, we have searched for the existence of an ACTH-independent pathway in an experimental model of a major parasitic disease like Chagas disease, in addition to characterizing potential alternative pathways of GC synthesis. To this end, C57BL/6 male mice were infected with T. cruzi (Tc), and evaluated throughout the acute phase for several parameters, including the kinetic of GC and ACTH release, the adrenal level of MC2R (ACTH receptor) expression, the p-PKA/PKA ratio as ACTH-dependent mechanism of signal transduction, as well as adrenal expression of IL-1β and its receptor, EPAC2 and PGE2 synthase. Our results reveal the existence of two phases involved in GC synthesis during Tc infection in mice, an initial one dealing with the well-known ACTH-dependent pathway, followed by a further ACTH-hyporesponsive phase. Furthermore, inflamed adrenal microenvironment may tune the production of intracellular mediators that also operate upon GC synthesis, like PGE2 synthase and EPAC2, as emerging driving forces for GC production in the advanced course of Tc infection. In essence, GC production seems to be associated with a biphasic action of PGE2, suggesting that the effect of PGE2/cAMP in the ACTH-independent second phase may be mediated by EPAC2. Fil: Barbosa, Esdras da Silva Oliveira. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina Fil: Roggero, Eduardo Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina Fil: González, Florencia Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina Fil: Fernández, Rocío del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina Fil: Carvalho, Vinicius Frias. Fundación Oswaldo Cruz; Brasil. Instituto Oswaldo Cruz; Brasil Fil: Bottasso, Oscar Adelmo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina Fil: Perez, Ana Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina Fil: Villar, Silvina Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Inmunología Clinica y Experimental de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Instituto de Inmunología Clinica y Experimental de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina

Glucocorticoid production in mammals is principally regulated by the action of the pituitary hormone adrenocorticotropin (ACTH) acting on its cognate membrane receptor on the zona fasciculata cells of the adrenal cortex. The receptor for ACTH consists of two essential components, a small seven transmembrane domain G protein-coupled receptor of the melanocortin receptor subgroup known as the melanocortin 2 receptor (MC2R) and a small single transmembrane domain protein that adopts a antiparallel homodimeric form and which is known as the melanocortin 2 receptor accessory protein (MRAP). MRAP is essential for the trafficking of the MC2R to the cell surface as well as being required for receptor responsiveness to ACTH at physiological concentrations – probably by facilitating ACTH binding, but possibly also by supporting G protein interaction with the MC2R. A number of studies have shown that ACTH stimulates the expression of functional receptor at the cell surface and the transcription of both MC2R and MRAP mRNA. However the time course of these transcriptional effects differs such that MRAP is expressed relatively rapidly whereas MC2R transcription responds much more slowly. Furthermore, recent data suggests that MRAP protein is turned over with a short half-life whereas MC2R has a significantly longer half-life. These findings imply that these two ACTH receptor proteins have distinct trajectories and that it is likely that MRAP-independent MC2R is present at the cell surface. In such a situation newly transcribed and translated MRAP could enable the rapid recruitment of functional receptor at the plasma membrane without the need for new MC2R translation. This may be advantageous in circumstances of significant stress in that the potentially complex and perhaps inefficient process of de novo MC2R translation, folding, post-translational modification and trafficking can be avoided.

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.

The low rate of cure of adrenocortical carcinomas (ACC) in children and adults is related to germ line TP53 mutation, late diagnosis, incomplete surgical resection, and lack of an efficient adjunctive therapy. To provide a new approach for the improvement of ACC diagnosis and therapy, the present study aimed to explicitly target ACC cells using gold nanoparticle (AuNP) probes bound to specific antibodies. Immunohistochemistry of ACC and positive and negative control tissue micro-sections under light microscopy was used to test a purified polyclonal antibody raised against the 80–93, outer loop 1 position of the human melanocortin receptor 2 (hMC2R). Both this and a control commercial antibody were found to specifically target cells known to express hMC2R. These were bound to FITC-labeled AuNPs and tested via direct immunofluorescence using the H295R ACC cell line. Both probes recognized only cells expressing hMC2R and exhibited very low background. Further studies are required to ascertain the potential of AuNPs bound to ACC cells for tumor diagnostics via imaging analysis or as a delivery device for targeted therapy.

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.