α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor.

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.

The Melanocortin System: A Promising Target for the Development of New Antidepressant Drugs.

Major depression is one of the most prevalent mental disorders, causing significant human suffering and socioeconomic loss. Since conventional antidepressants are not sufficiently effective, there is an urgent need to develop new antidepressant medications. Despite marked advances in the neurobiology of depression, the etiology and pathophysiology of this disease remain poorly understood. Classical and newer hypotheses of depression suggest that an imbalance of brain monoamines, dysregulation of the hypothalamic-pituitary-adrenal axis (HPAA) and immune system, or impaired hippocampal neurogenesis and neurotrophic factors pathways are cause of depression. It is assumed that conventional antidepressants improve these closely related disturbances. The purpose of this review was to discuss the possibility of affecting these disturbances by targeting the melanocortin system, which includes adrenocorticotropic hormone-activated receptors and their peptide ligands (melanocortins). The melanocortin system is involved in the regulation of various processes in the brain and periphery. Melanocortins, including peripherally administered non-corticotropic agonists, regulate HPAA activity, exhibit anti-inflammatory effects, stimulate the levels of neurotrophic factors, and enhance hippocampal neurogenesis and neurotransmission. Therefore, endogenous melanocortins and their analogs are able to complexly affect the functioning of those body's systems that are closely related to depression and the effects of antidepressants, thereby demonstrating a promising antidepressant potential.

Cigarette Smoke During Breastfeeding in Rats Changes Glucocorticoid and Vitamin D Status in Obese Adult Offspring.

Maternal smoking increases obesogenesis in the progeny. Obesity is associated with several hormonal dysfunctions. In a rat model of postnatal tobacco smoke exposure, we previously reported increased central fat depot and disruption of some hormonal systems in the adult offspring. As both glucocorticoids and vitamin D alter lipogenesis and adipogenesis, here we evaluated the metabolism of these two hormones in visceral adipose tissue (VAT) and liver by Western blotting, and possible associations with lipogenesis biomarkers in adult rats that were exposed to tobacco smoke during their suckling period. At postnatal day (PN) 3, dams and offspring of both sexes were exposed (S group) or not (C group) to tobacco smoke, 4 × 1 h/day. At PN180, corticosteronemia was lower in S male and higher in S female offspring, without alterations in peripheral glucocorticoid metabolism and receptor. Adrenal ACTH receptor (MC2R) was higher in both sexes of S group. Despite unchanged serum vitamin D, liver 25-hydroxylase was higher in both sexes of S group. Male S offspring had higher 1α-hydroxylase, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) in VAT. Both sexes showed increased ACC protein content and reduced sirtuin mRNA in liver. Male S offspring had lower liver peroxisome proliferator-activated receptor-α. Tobacco exposure during lactation induced abdominal obesity in both sexes via distinct mechanisms. Males and females seem to develop HPA-axis dysfunction instead of changes in glucocorticoid metabolism and action. Lipogenesis in VAT and liver, as well as vitamin D status, are more influenced by postnatal smoke exposure in male than in female adult rat offspring.

Adrenal gland plasticity in lactating rats and mice is sufficient to maintain basal hypersecretion of corticosterone.

Increased basal glucocorticoid secretion and a reduced glucocorticoid response during acute stress, despite only minor changes in the secretion of the major secretagogue adrenocorticotropic hormone (ACTH), have been documented in the peripartum period in several species. We recently showed that the adrenal gland, the site of glucocorticoid synthesis, undergoes substantial postpartum-associated plasticity in the rat at mid-lactation. Here, we asked the question whether adrenal changes already take place around parturition in the rat and in another species, namely the mouse. After demonstrating that several components of the adrenal machinery mediating cholesterol supply for steroidogenesis, including protein levels of hormone-sensitive lipase, low-density lipoprotein receptor (LDLR) and scavenger receptor class-B type-1 (SRB1), are upregulated, while hydroxymethylglutaryl coenzyme A reductase (HMGCR) is downregulated in the lactating rat one day after delivery, as previously observed at mid-lactation, we demonstrated profound changes in the mouse. In detail, protein expression of LDLR, SRB1, HMGCR and adrenal lipid store density were increased in the mouse adrenal one day after parturition as tested via western blot analysis and oil-red lipid staining, respectively. Moreover, using in vitro culture techniques, we observed that isolated adrenal explants from lactating mice secreted higher levels of corticosterone under basal conditions, but showed impaired responsiveness to ACTH, mimicking the in vivo scenario. These results suggest that mechanisms of adaptation in the maternal adrenal after delivery, namely increased cholesterol availability and decreased ACTH sensitivity, are crucial for the basal increase in circulating glucocorticoids and maternal stress hyporesponsiveness that are typical of this period.

Third transmembrane domain of the adrenocorticotropic receptor is critical for ligand selectivity and potency.

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, ß-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe(7) with D-Phe or D-naphthylalanine (D-Nal(2')) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe(7) with D-Nal(2') in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe(7) with D-Phe(7) in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe(7) in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.

Dynamic pituitary-adrenal interactions in response to cardiac surgery.

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 mechanisms underlying this change in a rat model of critical illness. CLINICAL STUDIES: Prospective observational study. ANIMAL STUDIES: Controlled experimental study. CLINICAL STUDIES: Cardiac surgery operating rooms and critical care units. ANIMAL STUDIES: University research laboratory. CLINICAL STUDIES: Twenty, male patients. ANIMAL STUDIES: Adult, male Sprague-Dawley rats. CLINICAL STUDIES: Coronary artery bypass graft-both on and off pump. ANIMAL STUDIES: Injection of either lipopolysaccharide or saline (controls) via a jugular vein cannula. CLINICAL STUDIES: Blood samples were taken for 24 hours from placement of the first venous access. Cortisol and adrenocorticotropic hormone were measured 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. Adrenocorticotropic hormone levels then returned toward preoperative values. Ultradian pulsatility of both adrenocorticotropic hormone and cortisol was maintained throughout the perioperative period in all individuals. The sensitivity of the adrenal gland to adrenocorticotropic hormone increased markedly at around 8 hours after surgery 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 sampling 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 measurement. 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, respectively. 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 reaction. In response to lipopolysaccharide, rats showed a pattern of adrenocorticotropic hormone and corticosterone that was similar to patients undergoing coronary artery bypass grafting. We were also able to demonstrate increased intra-adrenal corticosterone levels and an increase in steroidogenic acute regulatory protein, steroidogenic factor 1, and melanocortin type 2 receptor accessory protein messenger RNAs and steroidogenic acute regulatory protein, and a reduction in dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 and melanocortin type 2 receptor messenger RNAs, 6 hours after lipopolysaccharide injection. CONCLUSIONS: Severe inflammatory stimuli activate the hypothalamic-pituitary-adrenal axis resulting in increased steroidogenic activity in the adrenal cortex and an elevation of cortisol levels in the blood. Following coronary artery bypass grafting, there is a massive increase in both adrenocorticotropic hormone and cortisol secretion. Despite a subsequent fall of adrenocorticotropic hormone to basal levels, cortisol remains elevated and coordinated adrenocorticotropic hormone-cortisol pulsatility is maintained. This suggested that there is an increase in adrenal sensitivity to adrenocorticotropic hormone, which we confirmed in our animal model of immune activation of the hypothalamic-pituitary-adrenal axis. Using this model, we were able to show that this increased adrenal sensitivity results from changes in the regulation of both stimulatory and inhibitory intra-adrenal signaling pathways. Increased understanding of the dynamics of normal hypothalamic-pituitary-adrenal responses to major surgery will provide us with a more rational approach to glucocorticoid therapy in critically ill patients.

Alterations in the profile of blood neutrophil membrane receptors caused by in vivo adrenocorticotrophic hormone actions.

Elevated levels of adrenocorticotrophic hormone (ACTH) mobilize granulocytes from bone marrow into the blood, although these neutrophils are refractory to a full migratory response into inflamed tissues. Here, we investigated the dependence of glucocorticoid receptor activation and glucocorticoid-regulated protein annexin A1 (ANXA1) on ACTH-induced neutrophilia and the phenotype of blood neutrophil after ACTH injection, focusing on adhesion molecule expressions and locomotion properties. ACTH injection (5 µg ip, 4 h) induced neutrophilia in wild-type (WT) mice and did not alter the elevated numbers of neutrophils in RU-38486 (RU)-pretreated or ANXA1(-/-) mice injected with ACTH. Neutrophils from WT ACTH-treated mice presented higher expression of Ly6G⁺ANXA1(high), CD18(high), CD62L(high), CD49(high), CXCR4(high), and formyl-peptide receptor 1 (FPR1(low)) than those observed in RU-pretreated or ANXA1(-/-) mice. The membrane phenotype of neutrophils collected from WT ACTH-treated mice was paralleled by elevated fractions of rolling and adherent leukocytes to the cremaster postcapillary venules together with impaired neutrophil migration into inflamed air pouches in vivo and in vitro reduced formyl-methionyl-leucyl-phenylalanine (fMLP) or stromal-derived factor-1 (SDF-1α)-induced chemotaxis. In an 18-h senescence protocol, neutrophils from WT ACTH-treated mice had a higher proportion of ANXAV(low)/CXCR4(low), and they were less phagocytosed by peritoneal macrophages. We conclude that alterations on HPA axis affect the pattern of membrane receptors in circulating neutrophils, which may lead to different neutrophil phenotypes in the blood. Moreover, ACTH actions render circulating neutrophils to a phenotype with early reactivity, such as in vivo leukocyte-endothelial interactions, but with impaired locomotion and clearance.

Neuroendocrine tumor in liver with positive ACTH receptor: a case report.

A rare case of possible primary ectopic adrenocorticotropic hormone (ACTH)-producing tumor in the liver mimicking a liver hemangioma is reported. A 9-year-old boy, with Cushing syndrome, was referred for the assessment of ectopic ACTH-producing tumor. Ultrasound, CT scan, and MRI of the abdomen revealed a liver lesion suggestive of a hemangioma. (111)In-octreotide scintigraphy revealed focal activity in the liver, indicative of a somatostatin-positive lesion. (99m)Tc-labeled RBC scintigraphy was negative for hemangioma. After surgical resection of the tumor, the cortisol level converted to a normal range indicative of a rare possible primary ACTH-producing tumor in the liver, which was confirmed by histopathology.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion.

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

Glucocorticoid receptor and molecular chaperones in the pathogenesis of adrenal incidentalomas: potential role of reduced sensitivity to glucocorticoids.

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9ß) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9ß (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9ß, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

Leptin receptor antagonist treatment ameliorates the effects of long-term maternal hypoxia on adrenal expression of key steroidogenic genes in the ovine fetus.

We previously reported elevated adipose leptin expression, plasma leptin concentrations, and adrenocortical leptin receptor expression in the long-term hypoxic (LTH) ovine fetus. This study addressed whether leptin antagonist (LA) administration to LTH fetal sheep altered expression of key genes governing cortisol synthesis. Ewes were maintained at high altitude (3,820 meters) from 40 to 130 days gestation (dG), returned to Loma Linda University, and implanted with a maternal tracheal catheter. Reduced Po2 was maintained by nitrogen infusion. On 132 dG, LTH (n = 11) and age-matched, normoxic control (n = 11) fetuses underwent vascular catheter implantation. At 138 dG, fetuses were continuously infused with either saline or the LA (1.5 mg·kg(-1)·day(-1)) for 4 days and samples collected for blood gases, ACTH, and cortisol. Fetal adrenal cortex was collected for determination of steriodogenic acute regulatory protein (StAR), ACTH, and leptin receptor, cholesterol side-chain cleavage (CYP11A1), cytochrome P-450 11ß-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17ß-hydroxysteroid dehydrogenase (HSD3B) expression. In the saline-infused LTH fetuses, StAR, ACTH receptor, CYP11A1, and CYP17 expression was significantly lower compared with control (P < 0.05), whereas levels of CYP11B1, CYP21, and HSD3B mRNA were similar between groups. LA infusion restored expression of StAR, pSTAT3, CYP11A1, and CYP17, but not ACTH receptor, to normal ontogenic levels in the LTH group while having no effect on control fetuses. Neither fetal plasma ACTH nor cortisol concentrations were altered by LA infusion. We speculate that while leptin plays a role in governing expression of key enzymes and StAR in response to LTH, other factors play a role in modulating cortisol synthesis in these fetuses.

Control of CYP11B2/CYP11B1 expression ratio and consequences for the zonation of the adrenal cortex.

Access of corticotropin to glucocorticoid synthesis in adrenocortical cells is provided by the expression of the ACTH receptor (MC2R). Activation of the MC2R increases stimulatory G-protein, adenylyl cyclase, and protein kinase A (PKA) activities. Furthermore, PKA phosphorylates transcription factors that have a stimulating effect on glucocorticoid synthesis. Sensitivity of adrenocortical cells to renin/angiotensin-2 is conferred by the expression of the inhibitory G-protein-linked angiotensin-2 type 1 receptor (AT1R) that additionally associates to the phospholipase C-activating G-protein q. The AT1R is connected to the adrenal potassium sensory system and regulates calcium influx as well as phospholipase C-ß (PLC-ß) and thus calmodulin kinase-dependent transcription of steroidogenic enzymes. While AT1R signaling suppresses the influence of corticotropin on the generation of cyclic adenosine monophosphate, the expression of the AT1R and its associated enzyme activities are under the control of glucocorticoids. Thus, dominance of one of the two signaling pathways is dependent on two factors: the extracellular concentration of their ligands and the products of their signaling pathways. These findings are in favor of the hypothesis that the centripetal blood flow through the adrenal gland builds up a glucocorticoid gradient creating a morphogenetic field along which adrenal cortical cells adopt different functional states, leading to the typical zonation of the adrenal cortex.

The genetics of malignant melanoma: lessons from mouse and man.

Therapeutic resistance and proclivity for metastasis are hallmarks of malignant melanoma. Genetic, epidemiological and genomic investigations are uncovering the spectrum of stereotypical mutations that are associated with melanoma and how these mutations relate to risk factors such as ultraviolet exposure. The ability to validate the pathogenetic relevance of these mutations in the mouse, coupled with advances in rational drug design, has generated optimism for the development of effective prevention programmes, diagnostic measures and targeted therapeutics in the near future.

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Generation of glucocorticoid-producing cells derived from human pluripotent stem cells.

Adrenal insufficiency is a life-threatening condition resulting from the inability to produce adrenal hormones in a dose- and time-dependent manner. Establishing a cell-based therapy would provide a physiologically responsive approach for the treatment of this condition. We report the generation of large numbers of human-induced steroidogenic cells (hiSCs) from human pluripotent stem cells (hPSCs). Directed differentiation of hPSCs into hiSCs recapitulates the initial stages of human adrenal development. Following expression of steroidogenic factor 1, activation of protein kinase A signaling drives a steroidogenic gene expression profile most comparable to human fetal adrenal cells, and leads to dynamic secretion of steroid hormones, in vitro. Moreover, expression of the adrenocorticotrophic hormone (ACTH) receptor/co-receptor (MC2R/MRAP) results in dose-dependent ACTH responsiveness. This protocol recapitulates adrenal insufficiency resulting from loss-of-function mutations in AAAS, which cause the enigmatic triple A syndrome. Our differentiation protocol generates sufficient numbers of hiSCs for cell-based therapy and offers a platform to study disorders causing adrenal insufficiency.

Corticotropin releasing factor-1 receptor antagonism associated with favorable outcomes of male reproductive health biochemical parameters.

Background: Disruption in androgen profiles and testicular adrenal rest tumors in males with congenital adrenal hyperplasia (CAH) can negatively affect sexual activity and fertility. Adrenal hyperandrogenism suppresses gonadotropin secretion and testicular adrenal rest tumors (TARTS), despite being noncancerous lesions, cause obstructive azoospermia and impaired testosterone (T) production. Circulating T in men with uncontrolled CAH is often predominantly adrenal in origin, which is reflected in high androstenedione/testosterone ratios (A4/T). Therefore, decreased luteinizing hormone (LH) levels and an increased A4/T are markers of impaired fertility in these individuals. Methods: Oral tildacerfont 200 to 1000 mg once daily (QD) (n=10) or 100 to 200 mg twice daily (n=9 and 7) for 2 weeks (Study 201), and 400 mg QD (n=11) for 12 weeks (Study 202). Outcomes measured changes from baseline in A4, T, A4/T, and LH. Results: Mean T levels increased in Study 201 from 375.5 ng/dL to 390.5 ng/dL at week 2 (n=9), 485.4 ng/dL at week 4 (n=4) and 420.7 ng/dL at week 6 (n=4). In Study 202, T levels fluctuated in the normal range from 448.4 ng/dL at baseline to 412.0 ng/dL at week 12. Mean LH levels increased in Study 201 from 0.68 IU/L to 1.59 IU/L at week 2 (n=10), 1.62 IU/L at week 4 (n=5) and 0.85 IU/L at week 6 (n=4). In Study 202, mean LH levels increased from 0.44 IU/L at baseline to 0.87 IU/L at week 12. Mean A4/T decreased across both studies. In Study 201, the mean A4/T changed from a baseline of 1.28 to 0.59 at week 2 (n=9), 0.87 at week 4 (n=4), and 1.03 at week 6 (n=4). In Study 202, the A4/T decreased from baseline of 2.44 to 0.68 at week 12. Four men were hypogonadal at baseline; all experienced improved A4/T and 3/4 (75%) reached levels <1. Conclusion: Tildacerfont treatment demonstrated clinically meaningful reductions in A4 levels, and A4/T with concomitant increased LH levels indicating increased testicular T production. The data suggests improvement in hypothalamic-pituitary-gonadal axis function, but more data is required to confirm favorable male reproductive health outcomes.

Inflammation-induced lethargy is mediated by suppression of orexin neuron activity.

In response to illness, animals subvert normal homeostasis and divert their energy utilization to fight infection. An important and unexplored feature of this response is the suppression of physical activity and foraging behavior in the setting of negative energy balance. Inflammatory signaling in the hypothalamus mediates the febrile and anorectic responses to disease, but the mechanism by which locomotor activity (LMA) is suppressed has not been described. Lateral hypothalamic orexin (Ox) neurons link energy status with LMA, and deficiencies in Ox signaling lead to hypoactivity and hypophagia. In the present work, we examine the effect of endotoxin-induced inflammation on Ox neuron biology and LMA in rats. Our results demonstrate a vital role for diminished Ox signaling in mediating inflammation-induced lethargy. This work defines a specific population of inflammation-sensitive, arousal-associated Ox neurons and identifies a proximal neural target for inflammatory signaling to Ox neurons, while eliminating several others.

The beta-human chorionic gonadotropin-related peptide LQGV exerts anti-inflammatory effects through activation of the adrenal gland and glucocorticoid receptor in C57BL/6 mice.

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