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Breed differences exist between horses and ponies in circulating concentrations of several hormones, notably ACTH and insulin. These hormones regulate stress and metabolic responses, but in other species, they also impact leukocyte oxidant responses. The effects of these hormones on equine leukocytes have not been evaluated to date. If equine leukocytes are similarly regulated, breed differences in increased plasma hormone concentrations or altered sensitivity to them at the leukocyte level could result in breed-related differences in oxidant responses or oxidative status. The objective of this study was therefore to determine the effects of ex vivo exposure to adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), insulin, or leptin on reactive oxygen species (ROS) production from leukocytes isolated from horses and ponies. We hypothesized that ACTH, α-MSH, insulin, and leptin would alter oxidant responses from equine leukocytes in a breed specific manner. Blood was collected from 10 apparently healthy Quarter horses and seven Welsh ponies for isolation of neutrophils and peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation. Cells were incubated with media (negative control), microbial antigens (positive control), or ACTH, α-MSH, leptin, or insulin for two hours. Induced ROS production was quantified with a previously validated fluorometric assay. Data was compared within groups by comparing a stimulant within a group (horses or ponies) to baseline, between groups by comparing horse response to pony response, and among stimulants using one- and two-way, repeated measures ANOVA (P<0.05). There was no significant effect of breed on basal, microbial-induced, or hormone-induced ROS production from neutrophils (P=0.465) or PBMCs (P=0.749), but in neutrophils, a significant interaction between breed and stimulant was present (P=0.037). ROS production from PBMCs from horses after hormone exposure did not differ from cells exposed to media only (P=0.1520-0.8180). Similarly, neither leptin nor insulin exposure significantly induced ROS production from PBMCs from ponies (P= 0.2645 and 0.4678 respectively), but exposure to ACTH or α-MSH induced a significant increase in ROS production (P=0.0441 and 0.0440 respectively) compared to unstimulated cells. Hormones that vary in availability among breeds may induce ex vivo pro-oxidant responses in equine leukocytes, but specific effects are breed-, leukocyte type-, and hormone-dependent. Breed differences in hormonally induced leukocyte ROS production may warrant further investigation in the context of circulating oxidative stress and how this might relate to future disease risk., Competing Interests: Declaration of Competing Interest Dr. Hart has served as an unpaid consultant for and had travel expenses covered by Boehringer Ingelheim who produces a FDA labeled treatment for PPID in horses. None of the other authors have competing interests to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Porcine adrenocorticotrophic hormone (ACTH) has been considered valid for the ACTH stimulation test (ACTHST) in humans and dogs; however, its safety and efficacy for use in cats are unknown. Also, the equivalence between 5 µg/kg and 125 µg/cat dose of synthetic corticotropin (1-24 ACTH - cosyntropin/tetracosactide) is assumed for ACTHST in cats. This study evaluated the safety and effectiveness of different porcine recombinant ACTH doses for the ACTHST in healthy cats and its equivalence with tetracosactide. The study was divided into two arms. The first evaluated safety and equivalence of intravenous 1 µg/kg, 5 µg/kg, or 125 µg/cat porcine ACTH in seven healthy cats for the ACTHST evaluating basal and post-ACTH androstenedione, aldosterone, cortisol, and progesterone concentrations. In the second arm, the equivalence of the 125 µg/cat porcine ACTH dose was evaluated compared to results obtained using 125 µg/cat of tetracosactide in ten healthy cats regarding cortisol responses. In all tests, several cat-friendly strategies were adopted, and the ACTHST protocol involved basal and 60-minute post-ACTH blood sampling and intravenous ACTH injection. No adverse reactions were documented, and no tested cat showed any complications during the study. No porcine ACTH tested dose significantly increased androstenedione secretion. In contrast, all tested doses were able to increase progesterone concentration significantly (P < 0.05), and Δ-progesterone in response to 5 µg/kg or 125 µg/cat was considered equivalent (P > 0.99). The 125 µg/cat dose promoted greater responses for both cortisol and aldosterone, characterized by Δ-cortisol (P = 0.009) and Δ-aldosterone (P = 0.004). Despite equivalent Δ-cortisol results in response to 5 µg/kg or 125 µg/cat (P = 0.18); post-ACTH results of cortisol in response to 5 µg/kg only approximate statistical significance when compared with basal (P = 0.07). Porcine ACTH and tetracosactide significantly increased post-ACTH cortisol concentration (P < 0.0001) while the Δ-cortisol was slightly greater in response to the porcine ACTH (P = 0.006). These results suggest porcine ACTH could be an alternative source of corticotropin for the ACTHST in cats; however, maximum corticoadrenal stimulation seemed more reliable in response to a 125 µg/cat regarding cortisol and aldosterone., Competing Interests: Declaration of competing interest While preparing this work, the authors used the Grammar Check Service from Grammarly, Inc. (USA) to warrant correct spelling and grammar construction and for clarity and conciseness. After using this service, the authors reviewed and edited the content as needed and took full responsibility for the publication's content., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Directed differentiation of pluripotent stem cells into specialized cell types represents an invaluable tool for a wide range of applications. Here, we have exploited single-cell transcriptomic data to develop a stepwise in vitro differentiation system from mouse embryonic stem cells into adrenocortical cells. We show that during development, the adrenal primordium is embedded in an extracellular matrix containing tenascin and fibronectin. Culturing cells on fibronectin during differentiation increased the expression of the steroidogenic marker NR5A1. Furthermore, 3D cultures in the presence of protein kinase A (PKA)-pathway activators led to the formation of aggregates composed of different cell types expressing adrenal progenitor or steroidogenic markers, including the adrenocortical-specific enzyme CYP21A1. Importantly, in-vitro-differentiated cells responded to adrenocorticotropic hormone (ACTH) and angiotensin II with the production of glucocorticoids and mineralocorticoids, respectively, thus confirming the specificity of differentiation toward the adrenal lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthetic peptides have a wide range of clinical effects. Of particular interest are peptides based on adrenocorticotropic hormone (ACTH) both as already used and as potential drugs for preventing consequences of cerebral ischemia. However, it is necessary to study influence of the peptide on the brain cells under normal physiological conditions, including understanding the risks of their use. Here, we used high-throughput RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs) in the brain frontal cortex of rat receiving intraperitoneal administration of ACTH-like peptides ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP, or saline. We identified 258 and 228 DEGs, respectively, with the fold change &gt; 1.5 and Padj  &lt; 0.05 at 22.5 h after the first administration of Semax and ACTH(6-9)PGP. Metabolic pathways, characterizing both common and specific effects of the peptides on the transcriptome were identified. Both peptides predominantly caused decrease in expression of the genes associated with the immune system. At the same time, when comparing the effects of ACTH(6-9)PGP relative to Semax, DEGs were identified that characterized the main differences in the effects of the peptides. These genes were mostly downregulated and associated with neurosignaling systems and regulation of ion channels, thus characterizing differences in the effects of the peptides. Our data show how differences in the structure of ACTH derivatives are associated with the changes in the brain cell transcriptome following exposure to these related peptides. Furthermore, our results demonstrate that when studying influence of regulatory peptides on transcriptome under pathological conditions, it is necessary to take into account their actions under normal physiological conditions.

Pituitary tumors (PTs) represent about 10% of all intracranial tumors, and most are benign. However, some PTs exhibit continued growth despite multimodal therapies. Although temozolomide (TMZ), an alkylating chemotherapeutic agent, is a first-line medical treatment for aggressive PTs, some PTs are resistant to TMZ. Existing literature indicated the involvement of autophagy in cell growth in several types of tumors, including PTs, and autophagy inhibitors have anti-tumor effects. In this study, the expression of several autophagy-inducible genes, including Atg3, Beclin1, Map1lc3A, Map1lc3b, Ulk1, Wipi2, and Tfe3 in two PT cell lines, the mouse corticotroph AtT-20 cells and the rat mammosomatotroph GH4 cells were identified. Down regulation of Tfe3, a master switch of basal autophagy, using RNA interference, suppressed cell proliferation in AtT-20 cells, suggesting basal autophagy contributes to the maintenance of cellular functions in PT cells. Expectedly, treatment with bafilomycin A1, an autophagy inhibitor, suppressed cell proliferation, increased the cleavage of PARP1, and reduced ACTH production in AtT-20 cells. Treatment with two additional autophagy inhibitors, chloroquine (CQ) and monensin, demonstrated similar effects on cell proliferation, apoptosis, and ACTH production in AtT-20 cells. Also, treatment with CQ suppressed cell proliferation and growth hormone production in GH4 cells. Moreover, the combination of CQ and TMZ had an additive effect on the inhibition of cell proliferation in AtT-20 and GH4 cells. The additive effect of anti-cancer drugs such as CQ alone or in combination with TMZ may represent a novel therapeutic approach for PTs, in particular tumors with resistance to TMZ., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: The purpose of this study was to investigate the effects of escitalopram on the peripheral expression of hypothalamic-pituitary-adrenal (HPA) axis-related genes (FKBP51, HSP90, NR3C1 and POMC) and HPA-axis hormones in patients with panic disorder (PD)., Methods: Seventy-seven patients with PD were treated with escitalopram for 12 weeks. All participants were assessed for the severity of panic symptoms using the Panic Disorder Severity Scale (PDSS). The expression of HPA-axis genes was measured using real-time quantitative fluorescent PCR, and ACTH and cortisol levels were measured using chemiluminescence at baseline and after 12 weeks of treatment., Results: At baseline, patients with PD had elevated levels of ACTH and cortisol, and FKBP51 expression in comparison to healthy controls (all p < 0.01). Correlation analysis revealed that FKBP51 expression levels were significantly positively related to cortisol levels and the severity of PD (all p < 0.01). Furthermore, baseline ACTH and cortisol levels, and FKBP51 expression levels were significantly reduced after 12 weeks of treatment, and the change in the PDSS score from baseline to post-treatment was significantly and positively related to the change in cortisol (p < 0.01)., Conclusions: The results suggest that PD may be associated with elevated levels of ACTH and cortisol, and FKBP51 expression, and that all three biomarkers are substantially decreased in patients who have received escitalopram treatment., (© 2024. The Author(s).)

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nucleobindin-derived peptides, nesfatin-1 [NESF-1] and nesfatin-1-like-peptide [NLP] have diverse roles in endocrine and metabolic regulation. While both peptides showed a stimulatory effect on the synthesis of proopiomelanocortin (POMC), the adrenocorticotropic hormone (ACTH) precursor in mouse corticotrophs, whether NESF-1 and NLP have any direct effect on glucocorticoid [GC] synthesis in the adrenal cortex remains unknown. The main aim of this study was to determine if NESF-1 and/or NLP act directly on adrenal cortex cells to regulate cortisol synthesis in vitro. Whether NLP injection affects stress-hormone gene expression in the adrenal gland and pituitary in vivo in mice was also assessed. In addition, cortisol synthetic pathway in Nucb1 knockout mice was studied. Human adrenal cortical [H295R] cells showed immunoreactivity for both NUCB1/NLP and NUCB2/NESF-1. NLP and NESF-1 decreased the abundance of steroidogenic enzyme mRNAs, and cortisol synthesis and release through the AC/PKA/CREB pathway in H295R cells. Similarly, intraperitoneal injection of NLP in mice decreased the expression of enzymes involved in glucocorticoid (GC) synthesis in the adrenal gland while increasing the expression of Pomc, Pcsk1 and Crhr1 in the pituitary. Moreover, the melanocortin 2 receptor (Mc2r) mRNA level was enhanced in the adrenal gland samples of NLP injected mice. However, the global genetic disruption in Nucb1 did not affect most steroidogenic enzyme mRNAs, and Pomc, Pcsk2 and Crhr1 mRNAs in mice adrenal gland and pituitary gland, respectively. Collectively, these data provide the first evidence for a direct inhibition of cortisol synthesis and secretion by NLP and NESF-1. NUCB peptides might still elicit a net stimulatory effect on GC synthesis and secretion through their positive effects on ACTH-MC2R pathway in the pituitary., (© 2024. The Author(s).)

Objectives: A reduced adrenal reserve-associated cortisol production relative to the enhanced needs of chronic inflammation ( disproportion principle ) has been observed in rheumatoid arthritis (RA). We examined the possible clinical value of diurnal cortisol measurements in active RA on treatment response prediction., Methods: Diurnal cortisol production (measured at: 08-12:00/18:00-22:00) was assessed by electrochemiluminescence immunoassay in 28 consecutive patients with moderately/highly active RA, as well as 3 and 6 months after treatment initiation or/escalation. Twenty-eight COVID-19 patients and 28 age-matched healthy individuals (HC) served as controls., Results: Saliva diurnal cortisol production in patients with RA was similar to that of HC, despite 12-fold higher serum C reactive protein (CRP) levels, and lower than COVID-19 patients (area under the curve: RA: 87.0±37.6 vs COVID-19: 146.7±14.3, p<0.001), having similarly high CRP. Moreover, a disturbed circadian cortisol rhythm at baseline was evident in 15 of 28 of patients with RA vs 4 of 28 and 20 of 28 of HC and COVID-19 patients, respectively. Treatment-induced minimal disease activity (MDA) at 6 months was achieved by 16 of 28 patients. Despite comparable demographics and clinical characteristics at baseline, non-MDA patients had lower baseline morning cortisol and higher adrenocorticotropic hormone (ACTH) levels compared with patients on MDA (cortisol: 10.9±4.0 vs 18.4±8.2 nmol/L, respectively, p=0.005 and ACTH: 4.8±3.3 vs 2.4±0.4 pmol/L, respectively, p=0.047). Baseline morning cortisol <13.9 nmol/L predicted non-MDA at 6 months (75% sensitivity, 92% specificity, p=0.006). Prospective measurements revealed that individualised diurnal cortisol production remained largely unchanged from baseline to 3 and 6 months., Conclusions: An impaired adrenal reserve is present in patients with RA. Further studies to confirm that assessment of diurnal cortisol production may be useful in guiding treatment decisions and/or predicting treatment response in RA are warranted., Trial Registration Number: NCT05671627., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Conditions affecting the hypothalamic-pituitary-adrenal (HPA) axis are common in dogs. Testing the function of the HPA axis includes measurement of endogenous adrenocorticotropic hormone (eACTH) and performance of an adrenocorticotropic hormone (ACTH) stimulation test. Trazodone is commonly administered to dogs to decrease stress. In humans, trazodone significantly decreases plasma cortisol concentration via alpha-1 adrenergic activity., Objectives: Determine the influence of trazodone on eACTH and serum cortisol concentrations in healthy dogs., Animals: Fourteen healthy, adult, companion dogs., Methods: Prospective, randomized placebo-controlled study. Trazodone (8-10 mg/kg) or placebo was administered PO 1 hour before eACTH measurement and ACTH stimulation testing. After a ≥7-day wash-out period, dogs received the opposite treatment. Differences in eACTH, pre- and post-ACTH stimulation cortisol concentrations, and delta (difference between pre- and post-ACTH) cortisol concentrations were analyzed using a paired t or signed-rank test with a P < .05 significance level., Results: The eACTH concentrations were not significantly different (P = .23) between treatments. Similarly, no significant differences were found in the pre-ACTH cortisol concentrations between treatments (P = .40). Post-ACTH cortisol concentrations (P = .05) and delta cortisol concentrations (P = .04) were significantly lower when the dogs were treated with trazodone., Conclusions: Preliminary data suggest trazodone administration dampens the adrenocortical response to stimulation in healthy dogs. If similar effects are found in dogs with adrenal disease, the use of trazodone may affect diagnosis and clinical decision making in these populations., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Noise exposure induces metabolic disorders, in a latent, chronic and complex way. However, there is no direct evidence elucidating the relationship between low-intensity noise exposure and nonalcoholic fatty liver disease (NAFLD). Male mice (n = 5) on high-fat diet (HFD) were exposed to an average of 75 dB SPL noise for 3 months to reveal the effect of noise exposure on NAFLD, where the potential mechanisms were explored. In vivo (n = 5) and in vitro models challenged with dexamethasone (DEX) were used to verify the role of hypothalamus pituitary adrenal (HPA) axis activation in hepatic lipid metabolism. Typical chronic-restraint stress (CRS, n = 8) was used to explore the role of depression in modifying activity of HPA axis. Finally, animal experiment (n = 8) was repeated to validate the roles of depression and HPA axis activation in NAFLD development. Chronic low-intensity noise exposure exacerbated NAFLD in mice on HFD characterized by hepatocyte steatosis, modified lipid metabolism and inflammation level. Plasma ACTH in H + N group was 1.5-fold higher than that in HFD group. Transcription of glucocorticoid receptor target genes was increased by chronic low-intensity noise exposure in HFD-treated mice. Excessive glucocorticoids mimicking HPA axis activation induced NAFLD in vivo and in vitro. Plasma ACTH increase and lipid storage also occurred in depressive mice stressed by CRS. More interestingly, the same noise exposure simultaneously induced depression in mice, disrupted the HPA axis homeostasis and exacerbated NAFLD in a repeated experiment. Thus, three-month exposure to 75 dB SPL noise was sufficient to exacerbate NAFLD progress in mice, where activation of HPA axis played a critical role. Depression played an intermediate role and contributed to HPA axis activation up-stream of the exacerbation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In adrenal fasciculata cells stimulated by ACTH, Ca 2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca 2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca 2+ -containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca 2+ -free medium. ACTH rapidly increased the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in the normal medium, but did not influence [Ca 2+ ] i in the Ca 2+ -free medium, indicating that ACTH caused Ca 2+ influx into the cells. ACTH-induced Ca 2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca 2+ channel blocker but not by a T-type, N-type, or P-type Ca 2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca 2+ -free medium and slowly caused Ca 2+ influx into the cells. These results strongly suggest that Ca 2+ , as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

The pituitary represents an essential hub in the hypothalamus-pituitary-adrenal (HPA) axis. Pituitary hormone-producing cells (HPCs) release several hormones to regulate fundamental bodily functions under normal and stressful conditions. It is well established that the pituitary endocrine gland modulates the immune system by releasing adrenocorticotropic hormone (ACTH) in response to neuronal activation in the hypothalamus. However, it remains unclear how systemic inflammation regulates the transcriptomic profiles of pituitary HPCs. Here, we performed single-cell RNA-sequencing (scRNA-seq) of the mouse pituitary and revealed that upon inflammation, all major pituitary HPCs respond robustly in a cell type-specific manner, with corticotropes displaying the strongest reaction. Systemic inflammation also led to the production and release of noncanonical bioactive molecules, including Nptx2 by corticotropes, to modulate immune homeostasis. Meanwhile, HPCs up-regulated the gene expression of chemokines that facilitated the communication between the HPCs and immune cells. Together, our study reveals extensive interactions between the pituitary and immune system, suggesting multifaceted roles of the pituitary in mediating the effects of inflammation on many aspects of body physiology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Corticotropin-releasing hormone (CRH) is known for its crucial role in the stress response system, which could induce pituitary adrenocorticotropic hormone (ACTH) secretion to promote glucocorticoid release in the adrenal gland. However, little is known about other pituitary actions of CRH in teleosts. Somatolactin is a fish-specific hormone released from the neurointermediate lobe (NIL) of the posterior pituitary. A previous study has reported that ACTH was also located in the pituitary NIL region. Interestingly, our present study found that CRH could significantly induce two somatolactin isoforms' (SLα and SLβ) secretion and synthesis in primary cultured grass carp pituitary cells. Pharmacological analysis further demonstrated that CRH-induced pituitary somatolactin expression was mediated by the AC/cAMP/PKA, PLC/IP3/PKC, and Ca 2+ /CaM/CaMK-II pathways. Finally, transcriptomic analysis showed that both SLα and SLβ should play an important role in the regulation of lipid metabolism in primary cultured hepatocytes. These results indicate that CRH is a novel stimulator of somatolactins in teleost pituitary cells, and somatolactins may participate in the stress response by regulating energy metabolism.

Sleep deprivation is a common problem during pregnancy, but its impact on the fetus remains unclear. We aimed to investigate the effect of sleep deprivation during pregnancy on fetal outcomes and its mechanism in Sprague-Dawley rats. Sleep deprivation was performed from gestational day(GD) 1-19 using a multiplatform method for 18 h/day. Rats were sacrificed on GD20, and their blood and placentas were collected. Fetal and placental parameters were ascertained. Melatonin, adrenocorticotropic hormone (ACTH) and corticosterone were also measured in serum. The levels of arylalkylamine N-acetyltransferase (AANAT) and two melatonin receptors MT1 and MT2, in placental tissues were detected by western blotting. The inflammatory status and oxidative stress in serum and placentas were investigated. Miscarriage and intrauterine growth restriction were observed in the sleep deprivation group. Sleep deprivation resulted in an increased fetal absorption rate, while fetal weight, crown-rump length and placental weight were reduced. Placental histopathology showed that the labyrinth ratio in the sleep deprivation group was significantly reduced, with hypoplastic villi and obviously decreased blood vessels. Sleep deprivation decreased melatonin in serum and the expression of AANAT, MT1 and MT2 in placental tissues, elevated the oxidative stress products 8-hydroxy-deoxyguanosine (8-OHdG) and malondialdehyde(MDA) in serum and 4-hydroxynonenal (4HNE) in the placenta, and decreased the antioxidants superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum. Serum proinflammatory cytokines including interleukin-1-beta (IL-1β), interleukin-6 (IL-6), necrotizing factor-alpha (TNF-α), and interleukin-8(IL-8), were all elevated by sleep deprivation, and the inflammatory regulatory factor nuclear factor-κB p65 (NF-κB p65) in the placenta was enhanced when examined by immunohistochemistry. Corticosterone levels were comparable between the two groups, although ACTH levels were elevated significantly in the sleep deprivation group. Our study revealed that sleep deprivation during pregnancy can adversely impact fetal outcomes. Melatonin may play an important role in this pathology through the oxido-inflammatory process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of interests G.R.B. and S.R.B. have filed the patent application WO2023/083839 A1., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1-24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1-24) targeting residues within the H 6 F 7 R 8 W 9 and K 15 K 16 R 17 R 18 P 19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A 6-9 ) and KKRRP (A 15-19 ) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim was to evaluate the effect of ACTH on the mechanisms involved in peripheral blood mononuclear cells (PBMCs) infiltration into the ovary during dairy cattle proestrus. Regarding this, proper expression pattern of adhesion molecules must take place both in PBMCs and in endothelial cells. Argentinian Holstein cows (n = 12) were treated with 100 IU of ACTH every 12 h for 4 days before ovulation when ovariectomy was performed (day 18). Blood samples were taken on day 15 (0 h) and immediately before (72 h) and after (74 h) the last ACTH administration. In PBMCs, flow cytometry was performed to analyze CD44, CD11b and CD62-L expression along with gene expression of chemokines' receptors. Interleukin (IL)-4 and tumor necrosis factor-α (TNF-α) production was analyzed by flow cytometry after exposing PBMCs to autologous follicular fluid. In ovarian blood vessels, expression of the vascular endothelium cell adhesion-1 (VCAM-1) and the platelet endothelial cell adhesion molecule-1 was evaluated by immunohistochemistry. In T-lymphocytes, the expression of CD44 and CD11b was lower at 72 h in ACTH-treated cows (P < 0.05). In monocytes, the expression of CD11b and CD62-L was lower at 72 h in ACTH-treated cows (P < 0.05). Also, the percentage of IL-4 + cells was higher in ACTH-treated cows, meanwhile, the percentage TNF-α + cells was lower in ACTH-treated cows (P < 0.05). Finally, in the vessels associated with the preovulatory follicle VCAM-1 immunoexpression was lower in ACTH-treated cows (P < 0.05). Here, we present novel insights into the effect of stress during the preovulatory period on the inflammatory pathway necessary for ovulation., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Electroacupuncture (EA) can effectively reduce surgical stress reactions and promote postoperative recovery, but the mechanisms remain unclear. The present study aims to examine the effects of EA on the hyperactivity of the hypothalamic‒pituitary‒adrenal (HPA) axis and investigate its potential mechanisms. Male C57BL/6 mice were subjected to partial hepatectomy (HT). The results showed that HT increased the concentrations of corticotrophin-releasing hormone (CRH), corticosterone (CORT), and adrenocorticotropic hormone (ACTH) in the peripheral blood and upregulated the expression of CRH and glucocorticoid receptors (GR) proteins in the hypothalamus. EA treatment significantly inhibited the hyperactivity of the HPA axis by decreasing the concentration of CRH, CORT, and ACTH in peripheral blood and downregulating the expression of CRH and GR in the hypothalamus. Moreover, EA treatment reversed the HT-induced downregulation of oxytocin (OXT) and oxytocin receptor (OXTR) in the hypothalamus. Furthermore, intracerebroventricular injection of the OXTR antagonist atosiban blocked the effects of EA. Thus, our findings implied that EA mitigated surgical stress-induced HPA axis dysfunction by activating the OXT/OXTR signaling pathway., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hemp seed, the dried fruit of Cannabis sativa L. (Moraceae), has been extensively documented as a folk source of food due to its nutritional and functional value. This study evaluated the antidepressant effect of hemp seed oil (HSO) during its estrogen-like effect in Perimenopausal depression (PMD) rats induced by ovariectomy combined with chronic unpredictable mild stress (OVX-CUMS). Female SD rats (SPF, 10 weeks, sham operated group, ovariectomy (OVX) model group, ovariectomy - chronic unpredictable mild stress (OVX-CUMS) group, HSO + OVX-CUMS group, fluoxetine (FLU) + OVX-CUMS group, n=8) were subjected to treatment with HSO (4.32 g/kg) or fluoxetine (10 mg/kg) for 28 days (20 mL/kg by ig). Sucrose preference test (SPT), forced swimming test (FST), open field test (OFT), estrogen receptor α (ERα) and estrogen receptor β (ERβ) expression, estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), cortisol (CORT), adrenocorticotropic hormone (ACTH), corticotropin releasing hormone (CRH), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) levels are measured to evaluate the function of the hypothalamic-pituitary-ovarian (HPO) and hypothalamic-pituitary-adrenal (HPA) axis. The results showed that OVX-CUMS significantly decrease sucrose preference rate in SPT, increase immobility time in FST and OFT, and decrease movement distance and stand-up times in OFT. HSO treatment significantly improves depression-like behaviors, upregulates the expression of ERα and ERβ, improves HPO axis function by increasing E2 levels and decreasing FSH and LH levels, reverses HPA axis hyperactivation by decreasing CORT, ACTH, and CRH levels, and upregulates NE, 5-HT, and 5HIAA levels in model rats. The findings suggested that HSO could improve depression-like behavior in OVX-CUMS rats by regulating HPO/HPA axis function and neurotransmitter disturbance.

Pituitary pars intermedia dysfunction (PPID) is an age-related neurodegenerative disorder, affecting >20 % of older horses. There is a need for improved endocrine tests for early disease detection, and the thyrotropin-releasing hormone (TRH) stimulation test has been recommended for diagnosis of early or mild cases. However, it is currently not recommended for year-round use due to marked seasonal variability. The aims of this cohort study were to evaluate effects of month and season on adrenocorticotropic hormone (ACTH) responses to TRH stimulation and to derive monthly cut-offs for PPID diagnosis. Sixty-three horses were assigned to control (n = 17), subclinical PPID (n = 21) and clinical PPID (n = 25) groups, based on a composite reference standard that combined clinical history and examination findings with endocrine test results. TRH stimulation tests were performed monthly for a 12-month period. Circannual changes were evaluated with one- and two-way repeated-measures analysis of variance and receiver operating characteristic curve analysis was used to derive cut-off values for basal and TRH-stimulated ACTH. TRH-stimulated ACTH concentrations were lowest in February-May and highest in August-October. Specificity of both basal and 30 min post-TRH ACTH was generally higher than sensitivity, and TRH stimulation had improved diagnostic accuracy compared to basal ACTH, although its sensitivity was not significantly greater year-round. TRH stimulation tests yielded considerably more positive results than basal ACTH in the subclinical group, but few additional positive results in clinical PPID cases. There were large differences between cut-offs that maximised sensitivity or specificity for TRH-stimulated ACTH, highlighting the importance of considering clinical presentation alongside test results in diagnostic decision-making., Competing Interests: Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH 2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH 2 . Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species' reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Many patients with postural orthostatic tachycardia syndrome (POTS) are hypovolemic with plasma volume deficits of 10-30 %. Some also have low levels of aldosterone and diminished aldosterone-renin ratios despite elevations in angiotensin II, pointing to potential adrenal dysfunction. To assess adrenal gland responsiveness in POTS, we measured circulating levels of aldosterone and cortisol following adrenocorticotropin hormone (ACTH) stimulation., Methods: While on a low Na + diet (∼10 mEq/day), 8 female patients with POTS and 5 female healthy controls (HC) received a low dose (1 μg) ACTH bolus following a baseline blood sample. After 60 min, a high dose (249 μg) infusion of ACTH was administered to ensure maximal adrenal response. Venous aldosterone and cortisol levels were sampled every 30 min for 2 h., Results: Aldosterone increased in both groups in response to ACTH but was not different between POTS vs. HC at 60 min (53.5 ng/dL [37.8-61.8 ng/dL] vs. 46.1 ng/dL [36.7-84.9 ng/dL]; P = 1.000) or maximally (56.4 ng/dL [49.2-67.1 ng/dL] vs. 49.5 ng/dL [39.1-82.8 ng/dL]; P = 0.524). Cortisol increased in both groups in response to ACTH but was not different in patients with POTS vs. HC at 60 min (39.9 μg/dL [36.1-47.7 μg/dL] vs. 39.3 μg/dL [35.4-46.6 μg/dL]; P = 0.724) or maximally (39.9 μg/dL [33.9-45.4 μg/dL] vs. 42.0 μg/dL [37.6-49.7 μg/dL]; P = 0.354)., Conclusions: ACTH appropriately increased the aldosterone and cortisol levels in patients with POTS. These findings suggest that the response of the adrenal cortex to hormonal stimulation is intact in patients with POTS., Competing Interests: Declaration of competing interest CAS has served as a consultant for Lundbeck NA Ltd. and Theravance RSS. IB has served as a consultant for Lundbeck NA Ltd., Theravance RSS, Ammeal Pharmaceuticals, Regeneron, and Takeda Pharmaceutical. Patent holder of automated binder for treatment of Orthostatic Hypotension. RSS is a Cardiac Arrhythmia Network of Canada (CANet) Network Investigator. SRR is a consultant to Lundbeck LLC, Theravance Biopharma Inc., and Amneal Pharma related to neurogenic orthostatic hypotension; Consultant to Servier Affaires Medicales, Regeneron, argenx BV, and Antag Pharma related to postural orthostatic tachycardia syndrome. Honoraria from Spire Learning and Medscape for developing CME materials on neurogenic orthostatic hypotension. DMSB Chair for a Phase 2 study of an irritable bowel syndrome medication for Arena Pharmaceuticals with compensation. Past-President of the American Autonomic Society without financial compensation. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Bacterial sepsis is the leading cause of death in foals and is associated with hypothalamic-pituitary-adrenocortical axis (HPAA) dysfunction. HPAA function can be evaluated by an arginine-vasopressin (AVP) stimulation test., Hypotheses/objectives: Administration of AVP will stimulate a dose-dependent rise in systemic adrenocorticotropin-releasing hormone (ACTH) and cortisol in neonatal foals. There will be no response seen in corticotropin-releasing hormone (CRH) and baseline AVP will be within reference interval., Animals: Twelve neonatal foals, <72 hours old., Methods: HPAA function was assessed in foals utilizing 3 doses of AVP (2.5, 5, and 7.5 IU), administered between 24 and 48 hours of age in this randomized cross-over study. Cortisol, ACTH, CRH and AVP were measured at 0 (baseline), 15, 30, 60 and 90 minutes after AVP administration with immunoassays. The fold increase in cortisol and ACTH was calculated at 15 and 30 minutes compared to baseline., Results: All doses of AVP resulted in a significant increase in cortisol concentration over time, and a dose-dependent increase in ACTH concentration over time. ACTH and cortisol were significantly increased at 15 and 30 minutes, respectively after all 3 doses of AVP compared to baseline (P < .01). There was no change in endogenous CRH after stimulation with AVP., Conclusion and Clinical Importance: Administration of AVP is safe and results in a significant rise in ACTH and cortisol in neonatal foals. A stimulation test with AVP (5 IU) can be considered for HPAA assessment in septic foals., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H 6 F 7 R 8 W 9 ) and the "address" motif (K 15 K 16 R 17 R 18 P 19 ). Complete alanine substitution of the H 6 F 7 R 8 W 9 motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W 9  > R 8 , and substitution at F 7 and H 6 had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W 9  > R 8  = F 7 , (alanine substitution at H 6 was negligible). Complete alanine substitution at the K 15 K 16 R 17 R 18 P 19 motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cortisol is a neuroendocrine hormone of the hypothalamus-pituitary-adrenal (HPA) axis secreted from adrenal glands in response to stimulation by adrenocorticotropic hormone (ACTH) from the anterior pituitary and corticotropin releasing hormone (CRH) from the hypothalamus. Cortisol has multiple functionalities in maintaining bodily homeostasis - including anti-inflammatory influences - through its diurnal secretion pattern (which has been studied extensively); its secretion is also increased in response to major traumatic events such as surgery. Due to the adverse health consequences of an abnormal immune response, it is crucial to understand the effect of cortisol in modulating inflammation. To address this physiological issue, we characterize the secretion of cortisol using a high temporal resolution dataset of ten patients undergoing coronary arterial bypass grafting (CABG) surgery, in comparison with a control group not undergoing surgery. We find that cortisol exhibits different pulsatile dynamics in those undergoing cardiac surgery compared to the control subjects. We also summarize the causality of cortisol's relationship with different cytokines (which are one type of inflammatory markers) by performing Granger causality analysis.Clinical relevance- This work documents time-varying patterns of the HPA axis hormone cortisol in the inflammatory response to cardiac surgery and may eventually help improve patients' prognosis post-surgery (or in other conditions) by enabling early detection of an abnormal cortisol or inflammatory response and enabling patient specific remedial interventions.

Depression is a widespread disorder with a significant burden on individuals and society. There are various available treatments for patients with depression. However, not all patients respond adequately to their treatment. Recently, the opioid system has regained interest in depression studies. Research in animals and humans suggest that blocking the kappa opioid receptor (KOR) may potentially alleviate the symptoms of depression. The mechanism behind this effect is not fully understood. Stress and alterations in hypothalamic-pituitary-adrenal axis (HPA-axis) activity are thought to play a crucial role in depression. This study aimed to characterize stress hormones and stress-related protein expression following activation of KOR using a selective agonist. The longitudinal effect was investigated 24 h after KOR activation using the selective agonist U50,488 in Sprague Dawley rats. Stress-related hormones and protein expression patterns were explored using multiplex bead-based assays and western blotting. We found that KOR activation caused an increase in both adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in serum. Regarding protein assays in different brain regions, phosphorylated glucocorticoid receptors also increased significantly in thalamus (THL), hypothalamus (HTH), and striatum (STR). C-Fos increased time-dependently in THL following KOR activation, extracellular signal-regulated kinases 1/2 (ERK1/2) increased significantly in STR and amygdala (AMG), while phosphorylated ERK1/2 decreased during the first 2 h and then increased again in AMG and prefrontal cortex (PFC). This study shows that KOR activation alters the HPA axis and ERK signaling which may cause to develop mood disorders., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing financial or non-financial interests. KS and CS were employees at Alkermes Inc, USA at time of study completion., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Abstract: Evidence suggests an involvement of hypothalamic-pituitary-adrenal (HPA) axis dysregulation in the development and maintenance of fibromyalgia syndrome (FMS). However, studies on the stress response via the HPA-axis in individuals with FMS show conflicting results. To better understand the relationship between FMS and HPA-axis dysregulation, we (1) systematically summarized the current level of evidence on HPA biomarkers in individuals with FMS compared with individuals without and (2) evaluated whether FMS is associated with a specific pattern of HPA dysregulation. The main outcome measures were cortisol, adrenocorticotropic hormone (ACTH), corticotropin-releasing hormone (CRH), epinephrine, and norepinephrine. A systematic search of MEDLINE, EMBASE, and PsychMed yielded 47 studies eligible for meta-analysis, including 1465 individuals with FMS and 1192 FMS-free controls. No main effect of FMS was found on altered levels of blood cortisol, ACTH, CRH, and epinephrine. Compared with controls, salivary and urinary cortisol levels were decreased in individuals with FMS, whereas blood levels of norepinephrine were increased. However, heterogeneity of data was high with significant evidence for publication bias. Overall, the data are compatible with association of FMS with adrenocortical hypofunction in the presence of increased sympathetic tone. However, the data are partially contradictory, so it must be assumed that the data are highly dependent on the respective study designs, patient samples, and analytical methods and do not necessarily demonstrate an abnormal HPA-axis function in FMS., (Copyright © 2023 International Association for the Study of Pain.)

Ischemic stroke is an acute local decrease in cerebral blood flow due to a thrombus or embolus. Of particular importance is the study of the genetic systems that determine the mechanisms underlying the formation and maintenance of a therapeutic window (a time interval of up to 6 h after a stroke) when effective treatment can be provided. Here, we used a transient middle cerebral artery occlusion (tMCAO) model in rats to study two synthetic derivatives of adrenocorticotropic hormone (ACTH). The first was ACTH(4-7)PGP, which is known as Semax. It is actively used as a neuroprotective drug. The second was the ACTH(6-9)PGP peptide, which is elucidated as a prospective agent only. Using RNA-Seq analysis, we revealed hundreds of ischemia-related differentially expressed genes (DEGs), as well as 131 and 322 DEGs related to the first and second peptide at 4.5 h after tMCAO, respectively, in dorsolateral areas of the frontal cortex of rats. Furthermore, we showed that both Semax and ACTH(6-9)PGP can partially prevent changes in the immune- and neurosignaling-related gene expression profiles disturbed by the action of ischemia at 4.5 h after tMCAO. However, their different actions with regard to predominantly immune-related genes were also revealed. This study gives insight into how the transcriptome depends on the variation in the structure of the related peptides, and it is valuable from the standpoint of the development of measures for early post-stroke therapy.

Preterm infants experience multiple stressors including periodic neonatal hypoxia, maternal/caregiver separation, and acute pain from clinical procedures. Although neonatal hypoxia or interventional pain are associated with sexually dimorphic effects that may last into adulthood, the interaction of these common preterm stressors and caffeine pretreatment remains unknown. We hypothesize that an interaction of acute neonatal hypoxia, isolation, and pain modeling the experience of the preterm infant will augment the acute stress response and that caffeine routinely given to preterm infants will alter this response. Male and female rat pups were isolated and exposed to six cycles of periodic hypoxia (10% O 2 ) or normoxia (room air control) and/or intermittent pain by administering needle pricks (or touch control) to the paw on postnatal ( PD ) days 1-4 . An additional set of rat pups was pretreated with caffeine citrate (80 mg/kg ip) and studied on PD1 . Plasma corticosterone, fasting glucose, and insulin were measured to calculate homeostatic model assessment for insulin resistance (HOMA-IR) (index of insulin resistance). Glucocorticoid-, insulin-, and caffeine-sensitive gene mRNAs were analyzed in the PD1 liver and hypothalamus to evaluate downstream markers of glucocorticoid action. Acute pain with periodic hypoxia led to a large increase in plasma corticosterone, which was attenuated by pretreatment with caffeine. Pain with periodic hypoxia led to a 10-fold increase in hepatic Per1 mRNA expression in males, which was attenuated with caffeine. The augmentation of corticosterone and HOMA-IR at PD1 after periodic hypoxia with pain suggests early intervention to attenuate the stress response may mitigate the programming effects of neonatal stress.

Adrenal venous sampling (AVS) is thought to be the gold standard for primary aldosteronism (PA) subtype discrimination, during which the application of adrenocorticotropic hormone (ACTH) arouses heated debate. We aimed to identify the effect of ACTH on AVS and surgical outcomes. After propensity score matching (PSM), a total of 220 patients diagnosed with PA and completed AVS were included (110 without ACTH stimulation and 110 with ACTH stimulation). According to AVS results, surgeries were conducted in appropriate patients. ACTH stimulation significantly increased almost all selectivity index (SI) in both left adrenal vein (LAV) and right adrenal vein (RAV). We discovered that aldosterone/cortisol (A/C) value on dominant side significantly reduced after ACTH stimulation, with a reduction in lateralization index (LI) observed. Finally, 39 patients in unstimulated group and 32 patients in stimulated group completed surgery and enough follow-up. The comparison between surgical outcomes with and without ACTH stimulation was analyzed and the difference was not significant (p = .464). In conclusion, ACTH application significantly lowered A/C value instead of the relative aldosterone secretion index (RASI) value on dominant side, which did not yield superior surgical outcomes and might render confusing AVS interpretation., (© 2023 The Authors. The Journal of Clinical Hypertension published by Wiley Periodicals LLC.)

The central effectors of the stress system are greatly interconnected and include, among others, a large group of peptides derived from proopiomelanocortin. In addition to natural corticotropins, a number of artificial molecules that contain some ACTH fragments in their structure are also referred to members of this family. Some of them possess a wide range of biological activity. The molecular mechanism underlying the biological activity of such peptides is partly based on allosteric modulation of various receptors. We analyzed the ability of some biologically active synthetic corticotropins (ACTH(4-7)PGP, ACTH(6-9)PGP, ACTH(7-10)PGP), and glyproline PGPL to affect the GABA-receptor system of rat brain. The effects of the peptides were studied in the isolated plasma membranes of brain cells, as well as after systemic peptide administration in the rat model of acute restraint stress. The delayed effect of stress or preadministration of each of the studied peptides on [ 3 H]GABA binding was different for its high- and low-affinity-specific sites. The studied peptides individually affected the binding of [ 3 H]GABA in their own way. Acute restraint stress caused a decrease in [ 3 H]GABA binding at its low-affine site and did not affected the high-affine site. Preliminary peptide administration did not influence this effect of stress., (© 2023 John Wiley & Sons Ltd.)

Cushing's disease (CD) is a serious endocrine disorder attributed to an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that that subsequently leads to chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSC USP48 ) or USP8 (iPSC USP8 ) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSC USP48 and iPSC USP8 cultures, mifepristone induced a predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of Cushing's disease patients., Competing Interests: Conflicts of Interest All authors have read the journal's policy on disclosure of potential conflicts of interest.. Dr. Andreas G. Moraitis and Dr. Andrew E.Greenstein are employees and stock holders of Corcept. Dr. Kevin CJ Yuen has received research grants to the Barrow Neurological Institute from Crinetics, Ascendis, Corcept, and Amryt. Dr. Yuen has served as an advisory board member for Novo Nordisk, Ipsen, Amryt, Crinetics, Recordati and Xeris, and served as a speaker for Recordati, Novo Nordisk and Corcept. All other authors have no potential conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The effects of prenatal hypoxia on neurodevelopment are predominantly associated with impaired maternal glucocorticoid stimulation of the fetus, which is "imprinted" in altered sensitivity of glucocorticoid reception in brain structures of offspring and can affect brain plasticity during lifespan. This study aimed to investigate response of the brain glucocorticoid system to mild stress (MS) in adult rats that survived prenatal severe hypoxia (PSH) on embryonic days 14-16. In response to MS the control (but not PSH) rats demonstrate increased corticosterone levels, a decrease in exploratory activity and increased anxiety. In the raphe nuclei of adult PSH rats the expression of glucocorticoid receptors (GR) is increased without changes in serotonin levels in comparison with the control. MS induces a decrease in GR expression accompanied by up-regulation of tryptophan hydroxylase 2 (tph2) and down-regulation of monoamine oxidase A (maoa) transcription in the raphe nuclei of both control and PSH groups. PSH also causes significant deviations in GR expression and GR-dependent transcription in the hippocampus, the medial prefrontal cortex, but not in the amygdala of rats. However, in response to MS, PSH rats demonstrate mild changes in their activity, while in control animals the MS-induced activity of the glucocorticoid system in these brain structures is similar to intact PSH animals. Impaired activity of the glucocorticoid system in the extrahypothalamic brain structures of PSH rats is accompanied by increase in the hypothalamic corticotropin-releasing hormone (CRH) levels in comparison with the control regardless of MS. Synthesis of proopiomelanocortin (POMC) and release of adrenocorticotropic hormone (ACTH) into the blood are decreased in response to MS in the pituitary of control rats, which demonstrates a negative glucocorticoid feedback mechanism. Meanwhile, in the pituitary of PSH rats reduced POMC levels were found regardless of MS. Thus, prenatal hypoxia causes depression-like patterns in the brain glucocorticoid system with adverse reaction to mild stressors., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We evaluated the effect of ACTH 6-9 -Pro-Gly-Pro (ACTH 6-9 -PGP) peptide on the serum levels of pro- and anti-inflammatory cytokines in experimental animals under conditions of chronic restraint stress. Stress exposure over 2 weeks led to an increase in the levels of IL-1β, IL-6, and IFNγ in rats. Daily intraperitoneal injection of ACTH 6-9 -PGP in a dose of 5 μg/kg before stress exposure significantly decreased the levels of IL-6 and IFNγ by 4.8 and 49.3% respectively. Administration of the peptide in a dose of 50 μg/kg decreased the levels of IL-1β and IFNγ by 51.2 and 39.7%, respectively. However, no changes in the cytokine levels were observed after injection of the peptide in a dose of 500 μg/kg. Thus, ACTH 6-9 -PGP administered in doses of 5 and 50 μg/kg prevented the stress-induced changes in the levels of pro- and inflammatory cytokines., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Oxidative stress, arising from disrupted balance between reactive oxygen/nitrogen species (ROS/RNS) and antioxidant defences, has been implicated in the pathogenesis of stress-related disorders. There is a growing body of evidence that supports the relationship between the activity of the hypothalamic-pituitary-adrenal (HPA) stress system, oxidative stress and magnesium (Mg) homeostasis. The present study aimed to explore the gap in our current understanding of antigenotoxic and protective effects of Mg supplementation against excessive ROS production in male rats during chronic treatment with adrenocorticotropic hormone (ACTH). Our findings show that exposure to exogenous ACTH (10 μg/day, s.c., for 21 days), as one of the key mediators of the HPA axis and stress response, produced an increase in superoxide anion levels and a decrease in superoxide dismutase activity in plasma. We observed that Mg supplementation, starting seven days prior to ACTH treatment and lasting 28 days (300 mg/L of drinking water, per os), abolished these effects in experimental animals. Moreover, our study reveals that ACTH increased the susceptibility of peripheral blood lymphocytes to ex vivo H2O2-induced total and high-level oxidative DNA damage, while Mg completely reversed these effects. Collectively, these results highlight the promising role of Mg in stress-related conditions accompanied by increased oxidative stress in animals and support further investigation using human dietary trials.

Evidence shows that inflammatory responses may encompass the onset of severe depressive illness. Traditionally used licorice contains 18β-glycyrrhetinic acid (18βGA), which has been demonstrated to reduce inflammation and oxidative stress. This study investigates the antidepressant effects of 18βGA and the underlying mechanism in rats exposed to chronic unpredictable mild stress (CUMS). Wistar rats were exposed to CUMS for 36 consecutive days to establish depression. 18βGA (10, 20, and 50 mg/kg) or fluoxetine was given once daily (from day 30 to day 36). Thereafter, behavior parameters (sucrose preference test, forced-swimming test, open-field test, body weight), pro-inflammatory cytokines, neurotransmitters, adrenocorticotropic hormone (ACTH), corticosterone (CORT), and liver biomarkers were studied. Immunohistochemistry and western blot analyses were conducted to investigate the protein's expression. 18βGA (20 and 50 mg/kg) treatment increased sucrose intake, locomotion in the open-field test, decreased immobility time in the forced swim test, and improved body weight in CUMS-exposed rats. The therapy of 18βGA dramatically declined cytokines, ACTH and CORT and improved 5HT and norepinephrine in CUMS rats. Furthermore, BDNF and TrkB proteins were down-regulated in CUMS group, which was increased to varying degrees by 18βGA at doses of 20 and 50 mg/kg. Therefore, 18βGA ameliorates depressive-like behavior persuaded by chronic unpredictable mild stress, decreases neuroinflammation, liver biomarkers, stress hormones, and improves body weight, brain neurotransmitter concentration via activating on BDNF/TrkB signaling pathway in both PFC and hippocampus in rats., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To investigated the effects of suspended moxibustion stimulating Shenshu (BL23) and Guanyuan (CV4) acupoints on the amygdala and HPA axis in our rat model and elucidated the possible molecular mechanisms of moxibustion on kidney- deficiency symptom pattern (KYDS)., Methods: Sixty male Sprague Dawley rats were randomly divided into a control group ( 12) and an experimental group ( 48). Rats in the experimental group were given intramuscular injections of hydrocortisone to establish a KYDS model. The 48 rats successfully modeled were then randomly divided into a model group (model, 12), a carbenoxolone intraperitoneal injection group (CBX, 12), a moxibustion group (moxi, 12), and a moxi + CBX group ( 12). In the moxi, the Shenshu (BL23) and Guanyuan (CV 4) acupoints were treated with moxibustion for 14 d. After treatment, measures were taken of serum levels of corticosterone (CORT), adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH). The expression of mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs), 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1), CRH, and ACTH in the rats' amygdala, hypothalamus, or pituitary (as appropriate) was detected. Data were analyzed using one-way analysis of variance., Results: Compared with those of the control group, the serum levels of CRH, ACTH, and CORT; the mRNA and protein expressions of MR, GR, and 11β-HSD1 in the amygdala; the mRNA and protein expressions of 11β-HSD1 in the hypothalamus; the CRH mRNA expression in the amygdala and hypothalamus; and the ACTH mRNA expression in the pituitary of the rats in the model group were all significantly decreased (0.05 or 0.01). After treatment with moxibustion, all the aforementioned observation indices except for 11β-HSD1 mRNA expression were ameliorated compared with those in the model group (0.05 or 0.01)., Conclusions: Suspended moxibustion can effectively improve the serum levels of ACTH, CRH, and CORT and can up-regulate the mRNA and protein expressions of MR, GR, 11β-HSD1, CRH, and ACTH in the amygdala and hypothalamus of KYDS rats. This may be one of the molecular mechanisms with which moxibustion alleviates KYDS.

Winter and spring precipitation are predicted to increase in the Midwest region of the United States, causing muddy conditions. In a previous experiment, Angus cows (8 per treatment) were paired based on initial body weight (BW) and one cow from each pair was randomly allocated to either the mud or control treatment. Though cows consumed the same amount of dry matter, cows in the mud treatment weighed 37.4 kg less than cows in the control treatment by day 269 of gestation. The objective of this experiment was to evaluate developmental programming effects of steers born to cows in the mud treatment (MUD; n = 7) or the control treatment (CON; n = 6). Steers were weighed at birth and then weekly from approximately 56 d of age until weaning and were subjected to a glucose tolerance test (GTT) and adrenocorticotropic hormone (ACTH) challenge after weaning. Steers were then placed in the feedlot for an 84-d growing phase and were weighed weekly and 12th rib back fat (BF) and ribeye area (REA) were imaged every 28 d using ultrasonography. Data were analyzed as a randomized complete block design with repeated measurements when appropriate (SAS 9.4). Although there was a 37.4 kg decrease in BW of cows by the end of gestation, there was no evidence of a pen treatment effect on calf birth weight (P = 0.60) or weaning weight (P = 0.99). Additionally, there was no evidence of a pen treatment × day effect for steer BW from birth to weaning (P = 0.67) or growing phase BW (P = 0.60). There was evidence of a treatment × day of growing phase effect (P = 0.02) for BF, such that CON steers had greater BF on day 28 of the growing phase; however, there was no evidence of a treatment × day effect for REA (P = 0.20). Furthermore, there was no evidence of a pen treatment effect for the growing phase average daily gain (P = 0.74), dry matter intake (P = 0.65), gain:feed (P = 0.48), plasma glucose concentration (P = 0.67) or plasma insulin concentration (P = 0.61) in response to the GTT, or plasma cortisol concentration in response to the ACTH challenge (P = 0.51). These results indicate that while mud increased net energy requirements for cows in the MUD treatment, there were no subsequent effects observed for steer BW, gain:feed, or response to glucose and ACTH during the growing phase., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C-C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The sensitivity of the adrenocorticotropic hormone (ACTH) stimulation test to detect Cushing's Syndrome (CS) using a depot formulation needs to be evaluated. The aims of this study were to propose a reference interval (RI) for cortisol values 1-hour after administration of a low-dose of depot ACTH in healthy dogs, and to evaluate the sensitivity of this test to detect CS, differentiating among types of CS based on ultrasound findings. Forty-one healthy dogs (20 males, 21 females) were prospectively included. Additionally, 90 dogs with CS (31 males, 59 females) were retrospectively included. Dogs with CS were ultrasonographically classified as follows: 44 dogs with symmetrical adrenomegaly consistent with pituitary-dependent hypercortisolism (PDH), 8 dogs with unilateral adrenomegaly and atrophy of the contralateral adrenal gland or unilateral or bilateral adrenomegaly with malignancy features consistent with adrenal-dependent hypercortisolism (ADH), 34 dogs with equivocal adrenal asymmetry (EAA) and 4 dogs with normal adrenal thickness. In healthy dogs, lower and upper limit of the 95% RI for 1-hour post-ACTH cortisol concentration and their 90% confidence intervals, were 4.4 (2.7-5.8) μg/dl and 18.4 (16.5-20.0) μg/dl, respectively. Post-ACTH cortisol concentration was above the RI in 90.0% (ci95%, 76.1-100) of dogs with CS. An elevated post-ACTH cortisol concentration was detected in 95.5% (ci95%, 76.1-100) of dogs with PDH, 62.5% (ci95%, 46.1-78.9) of dogs with ADH and 88.2% (ci95%, 69.1-100) of dogs with EAA. The sensitivity of the ACTH stimulation test using a low-dose of depot ACTH in high in dogs with CS., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Ltd.)

Previous studies show that stressful events after ovulation in sows significantly impaired the embryo cleavage with a significant elevation of blood cortisol. However, the effects of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol on fertilization and embryo development remain to be specified, and whether they damage pig embryos directly or indirectly is unclear. This study demonstrated that embryo development was unaffected when pig parthenotes were cultured with different concentrations of CRH/ACTH/cortisol. However, embryo development was significantly impaired when the embryos were cocultured with pig oviductal epithelial cells (OECs) in the presence of CRH/cortisol or cultured in medium that was conditioned with CRH/cortisol-pretreated OECs (CRH/cortisol-CM). Fertilization in CRH/cortisol-CM significantly increased the rates of polyspermy. CRH and cortisol induced apoptosis of OECs through FAS and TNFα signaling. The apoptotic OECs produced less growth factors but more FASL and TNFα, which induced apoptosis in embryos. Pig embryos were not sensitive to CRH because they expressed no CRH receptor but the CRH-binding protein, and they were tolerant to cortisol because they expressed more 11-beta hydroxysteroid dehydrogenase 2 (HSD11B2) than HSD11B1. When used at a stress-induced physiological concentration, while culture with either CRH or cortisol alone showed no effect, culture with both significantly increased apoptosis in OECs. In conclusion, CRH and cortisol impair pig fertilization and preimplantation embryo development indirectly by inducing OEC apoptosis via the activation of the FAS and TNFα systems. ACTH did not show any detrimental effect on pig embryos, nor OECs.

We studied the biological effect of chronic exposure to multifrequency electromagnetic fields simulating the effects of 5G NR/IMT-2020 mobile communication systems. Male Wistar rats were exposed to 24-h radiation (250 μW/cm 2 ) for 4 months. The exploratory activity of the animals and blood concentrations of ACTH and corticosterone were evaluated at the end of each month of exposure and 1 month after exposure. The results suggest that exposure to multifrequency electromagnetic field simulating the effects of 5G systems affected functional activity of the hypothalamus-pituitary-adrenal axis and was stressful in nature., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

This research paper addresses the hypothesis that stress, induced by ACTH administration and cortisol release increases somatic cell count (SCC) in mammary secretion, and improves the effectiveness of dry off in goats. We report indicators of milk synthesis and mammary gland involution during dry off. Thirty Saanen goats were subjected to abrupt dry off and treatments: (1) ACTH administration (ACTH) or (2) placebo (Control) on days 1, 3, 6, 9, 12, 15, 30, and 60 of dry off. The expression of target genes in mammary tissue that are related to milk synthesis and cell survival such as insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositol-3-kinase (PIK3CA), protein kinase B (AKT1) and mechanistic target of rapamycin (MTOR), casein (CSN2), lactalbumin (LALBA) and lactoferrin (LF) were evaluated, and plasma cortisol concentration, SCC, leucocyte count, and microbiological analyses in milk and mammary secretions were assessed. ACTH significantly downregulated the expression of IGF1R and upregulated the expression of PIK3CA in mammary tissue, increased lactoferrin concentration and SCC, and changed immune cell levels in mammary secretions compared to Control. Furthermore, ACTH administration increased the percentage of dry goats compared to the Control (73 vs. 46%, respectively). We conclude that the effect of stress via ACTH administration and cortisol release accelerated mammary involution during the early dry-off period.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have crucial roles in regulating energy homeostasis. The melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) have been shown to regulate neural MCRs in a species-specific manner. The potential effects of MRAP1 and MRAP2 on canine neural MCRs have not been investigated before. Herein, we cloned canine (c) MC3R and identified one canine MRAP2 splice variant, MRAP2b , with N-terminal extension of cMRAP2a. Canine MC3R showed higher maximal responses to five agonists than those of human MC3R. We further investigated the modulation of cMRAP1, cMRAP2a, and cMRAP2b, on cMC3R and cMC4R pharmacology. For the cMC3R, all MRAPs had no effect on trafficking; cMRAP1 significantly decreased B max whereas cMRAP2a and cMRAP2b significantly increased B max . Both MRAP1 and MRAP2a decreased R max s in response to α-MSH and ACTH; MRAP2b only decreased α-MSH-stimulated cAMP generation. For the MC4R, MRAP1 and MRAP2a increased cell surface expression, and MRAP1 and MRAP2a increased B max s. All MRAPs had increased affinities to α-MSH and ACTH. MRAP2a increased ACTH-induced cAMP levels, whereas MRAP2b decreased α-MSH- and ACTH-stimulated cAMP production. These findings may lead to a better understanding of the regulation of neural MCRs by MRAP1 and MRAP2s.

Objective: This study served to compare the degree of adrenocortical suppression following a 2-week administration of loteprednol etabonate (LE) and prednisolone acetate (PA) ophthalmic drops., Procedures: In this prospective double-masked triple-crossover study, 21 clinically healthy dogs were randomized to receive loteprednol etabonate ophthalmic suspension 0.5%, prednisolone acetate ophthalmic suspension 1%, or artificial tears (AT). Each group (LE, PA, and AT) received one drop in each eye every 12 h for 2 weeks, followed by a 3-week washout period between treatment blocks. ACTH stimulation tests were performed before and after each treatment block. Serum cortisol samples were drawn before and 60 min after administration of 1 μg/kg cosyntropin IV. Repeated-measurement ANOVA followed by a Tukey's multiple comparisons test (or a Friedman test followed by a Dunn's multiple comparisons test) were used to compare pre- and post-treatment cortisol values between each group. A p-value of ≤.05 was considered significant., Results: A total of 18 dogs completed the study. Prestimulation cortisol values were lower in the PA group compared to the LE (p = .0106), but not AT (p = .0589) groups, and post-stimulation cortisol values were lower in the PA group than either LE (p = .0005) or AT (p = .0002) groups. There was no significant difference detected in pre- or post-stimulation cortisol values after the treatment periods between LE and AT., Conclusions: Based on the reduced suppression of cortisol values, LE caused significantly less hypothalamic-pituitary-adrenal axis suppression than PA. A topical steroid with minimal adrenocortical suppression, such as LE, may be favorable in patients where systemic glucocorticoid effects should be avoided., (© 2022 American College of Veterinary Ophthalmologists.)

In bony vertebrates, melanocortin 2 receptor (Mc2r) specifically binds adrenocorticotropic hormone (ACTH) and is responsible for mediating anterior pituitary signaling that stimulates corticosteroid production in the adrenal gland/interrenal cells. In bony fishes Mc2r requires the chaperoning of an accessory protein (Mrap1) to traffic to the membrane surface and bind ACTH. Here, we evaluated the structure and pharmacological properties of Mc2r from the Senegal bichir (Polypterus senegalus), which represents the most basal bony fish from which an Mc2r has been pharmacologically studied to date. In our experiments, cDNA constructs of the Mc2r from the Senegal bichir (sbMc2r) and various vertebrate Mrap1s were heterologously co-expressed in Chinese hamster ovary (CHO) cells, stimulated by ACTH or melanocyte-stimulating hormone (α-MSH) ligands, and assessed using a luciferase reporter gene assay. When expressed without an Mrap1, sbMc2r was not activated by ACTH. When co-expressed with Mrap1 from either chicken (Gallus gallus) or bowfin (Amia calva), sbMc2r could be activated in a dose-dependent manner by ACTH, but not α-MSH. Co-expression of sbMrap2 with sbMc2r resulted in no detectable activation of the receptor. Collectively, these results demonstrate that sbMc2r has pharmacological properties similar to those of Mc2rs of later-evolved bony fishes, such as Mrap1 dependence and ACTH selectivity, indicating that these qualities of Mc2r function are ancestral to all bony fish Mc2rs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Male and female rats were exposed to repeated restraint to determine how changes in serotonin (5-hydroxytryptamine; 5-HT) 1A receptors associate with stress hypothalamic-pituitary-adrenal (HPA) axis habituation., Methods: In response to 2-hour episodes of restraint, repeated daily for 5 consecutive days, males and females displayed reliable declines in HPA output, indicated by diminished adrenocorticotropin and corticosterone secretory responses. Using the 5-HT 1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) as a pharmacological challenge for inducing hypothermia and elevations in plasma corticosterone, males displayed sensitized hypothermal responses after repeated restraint, whereas corticosterone responses to 8-OH-DPAT were enhanced in both sexes following single or repeated exposure., Results: Only males showed elevations in 5-HT 1A receptor G-protein coupling responses in the dorsal raphe after repeated restraint, whereas only females showed an increase in 5-HT 1A receptor responses in the hippocampus following single or repeated exposure. G-protein coupling responses within both regions correlated positively with 5-HT 1A receptor binding capacity. Thus, despite expressing similar capacities for stress HPA axis habituation, males and females emerged from repeated restraint to show region-specific changes in 5-HT 1A receptor function that may be explained, at least in part, by changes in receptor availability., Conclusions: Based on the hypothermal and corticosteroid responses to 8-OH-DPAT, the present data suggest that stress habituation is met by an increase in the sensitivity of presynaptic 5-HT 1A receptors in males and by an increase in the sensitivity of a population of postsynaptic receptors in both sexes., (© The Author(s) 2022. Published by Oxford University Press on behalf of CINP.)