ACTH Concentrations Research Articles

Diurnal Range and Intra-patient Variability of ACTH Is Restored With Remission in Cushing's Disease.

Single ACTH measurements have limited ability to distinguish patients with Cushing's disease (CD) from those in remission or with other conditions. To investigate the changes in ACTH levels before and after transsphenoidal surgery (TSS) to identify trends that could confirm remission from CD and help establish ACTH cutoffs for targeted clinical trials in CD. Retrospective analysis of CD patients who underwent TSS from 2005 to -2019. Referral center. CD patients (n = 253) with ACTH measurements before and after TSS. TSS for CD. Remission after TSS. Remission was observed in 223 patients after TSS. Those in remission had higher ACTH variability at AM (P = .02) and PM (P < .001) time points compared to nonremission. The nonremission group had a significantly narrower diurnal range compared to the remission group (P = <.0001). A decrease in plasma ACTH of ≥50% from mean preoperative levels predicted CD remission after TSS, especially when using PM values. The absolute plasma ACTH concentration and ratio of preoperative to postoperative values were significantly associated with nonremission after multivariable logistic regression (adj P < .001 and .001, respectively). Our findings suggest that ACTH variability is suppressed in CD, and remission from CD is associated with the restoration of this variability. Furthermore, a decrease in plasma ACTH by 50% or more may serve as a predictor of remission post-TSS. These insights could guide clinicians in developing rational outcome measures for interventions targeting CD adenomas.

Crinecerfont, a CRF1 Receptor Antagonist, Lowers Adrenal Androgens in Adolescents With Congenital Adrenal Hyperplasia.

Crinecerfont, a corticotropin-releasing factor type 1 receptor antagonist, has been shown to reduce elevated adrenal androgens and precursors in adults with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD), a rare autosomal recessive disorder characterized by cortisol deficiency and androgen excess due to elevated adrenocorticotropin. To evaluate the safety, tolerability, and efficacy of crinecerfont in adolescents with 21OHD CAH. This was an open-label, phase 2 study (NCT04045145) at 4 centers in the United States. Participants were males and females, 14 to 17 years of age, with classic 21OHD CAH. Crinecerfont was administered orally (50 mg twice daily) for 14 consecutive days with morning and evening meals. The main outcomes were change from baseline to day 14 in circulating concentrations of ACTH, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone. 8 participants (3 males, 5 females) were enrolled; median age was 15 years and 88% were Caucasian/White. After 14 days of crinecerfont, median percent reductions from baseline to day 14 were as follows: ACTH, -57%; 17OHP, -69%; and androstenedione, -58%. In female participants, 60% (3/5) had ≥50% reduction from baseline in testosterone. Adolescents with classic 21OHD CAH had substantial reductions in adrenal androgens and androgen precursors after 14 days of oral crinecerfont administration. These results are consistent with a study of crinecerfont in adults with classic 21OHD CAH.

Changes in the functional activity of the hypothalamic-pituitaryadrenal axis in rats of both sexes with Guerin’s carcinoma against the background of hypothyroidism

The hypothalamic-pituitary-adrenal axis (HPA) makes the body’s response to various stress factors, interacting with the thyroid and gonadal axis. The aim of our work was to study the effect by induced hypothyroidism on changes in the functioning of the HPA axis in rats with Guerin’s carcinoma of both sexes. Materials and methods. The experiment was performed in white outbred rats of both sexes. Hypothyroidism was induced in animals by medication for 30 days with Mercazolil, and then Guerin’s carcinoma was transplanted (the main group). The reference groups covered animals with the independent growth of Guerin’s carcinoma and with independent hypothyroidism, as well as intact animals (the normal conditions). On day 18 of the tumor growth, the animals were sacrificed, and, using standard ELISA kits, in the serum and the homogenates of the adrenal glands, the tumor and the perifocal zone, the level of cortisol was determined; in the homogenates of the hypothalamus we measured the concentration of CT-releasing hormone (CRH) and in the pituitary gland the concentration of ACTH. Results. Induced hypothyroidism both in males and females caused adrenal insufficiency. The growth of Guerin’s carcinoma increased the level of CT-releasing hormone in males by 9.6 times and cortisol in the adrenal glands in females and males by 1.3 times and 2.3 times (p&lt;0.05), respectively. In the main group females and males we revealed a decrease in the level of CT-releasing hormone of the hypothalamus by an average of 1.4 times, ACTH in the pituitary gland by 1.5-1.8 times, but an increase in the cortisol concentration in blood and the adrenal glands only in males by an average of 1.5 times (p˂0.05). Only in males of the main group in the tumor and its perifocal zone, the content of cortisol increased by 5.9 times and 1.6 times (р˂0.05), respectively. Conclusion. The HPA axis has a gender-specific response to the growth of a malignant tumor, both in its independent growth variant and against the background of hypothyroidism, which in its independent variant causes adrenal insufficiency.

Analysis of Immunobiochemical Parameters in Overweight People in Assessing the Risk of Cardiovascular Diseases

A comparative analysis of specific immunobiochemical parameters, including natural antibodies (NAb) to endogenous regulators of the cardiovascular system, adrenal and gastrointestinal hormones, was performed in students aged 18-22 years with normal and increased body weight (the body mass index from 18.5 to 24.9 kg/m2 and from 25 to 29.9 kg/m2, respectively). The serum content of NAb and hormones was determined by ELISA. The level of the studied indicators depended on the body mass index value. In overweight subjects, the main immune indicators of the biogenic amine system, renin-angiotensin system, and kinin system exceeded the normal. The cortisol level was higher than in subjects with normal body weight. Aldosterone secretion was less dependent on the ACTH content and was lower than in students with normal body weight. The content of cholecystokinin and gastrin corresponded to the values for overweight. These trends in hormone contents are a predisposing factor for further weight gain. Practical significance of the combined assessment of disturbances in the immunological and biochemical homeostasis has been established. Analysis of the adrenal and gastrointestinal hormones can predict the risk of weight gain, but at the same time, changes in the level of immunological indicators in subjects with increased body weight characterizes the possibility of developing cardiovascular pathologies.

A Prospective Analysis of the Metyrapone Short Test Using Targeted and Untargeted Metabolomics

Introduction: The present study aimed to prove the metyrapone short test in a day clinic to be suitable for examining the integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with suspected secondary and tertiary adrenal insufficiency and to identify novel effector molecules in acute stress response. Methods: 44 patients were prospectively enrolled. Based on stimulated 11-deoxycortisol levels, patients were divided into a physiological (11-deoxycortisol ≥70 μg/L) and a pathological (11-deoxycortisol <70 μg/L) response group. Clinical follow-up examination was performed for validation. Ultraperformance liquid chromatography tandem mass spectrometry and a Fourier-transform-ion-cyclotron-resonance-mass-spectrometry were used for targeted and untargeted steroid metabolomics. Results: At baseline, lower levels of cortisone (42 vs. 50 nmol/L, p = 0.048) and 17-OH-progesterone (0.6 vs. 1.2 nmol/L, p = 0.041) were noted in the pathological response group. After metyrapone administration, the pathological response group exhibited significantly lower 11-deoxycortisol (39.0 vs. 94.2 μg/L, p < 0.001) and ACTH (49 vs. 113 pg/mL, p < 0.001) concentrations as well as altered upstream metabolites. Untargeted metabolomics identified a total of 76 metabolites to be significantly up- or downregulated by metyrapone. A significant increase of the bile acid glycochenodeoxycholic acid (GCDC, p < 0.01) was detected in both groups with an even stronger increase in the physiological response group. After a mean follow-up of 17.2 months, an 11-deoxycortisol cut-off of 70 µg/L showed a high diagnostic performance (sensitivity 100%, specificity 96%). Conclusion: The metyrapone short test is safe and feasible in a day clinic setting. The alterations of the bile acid GCDC indicate that the liver might be involved in the acute stress response of the HPA axis.

Sensitivity of Different ACTH and Cortisol Concentration Values in Corticotropin-Releasing Hormone Based Tests in Cushing’s Disease

ABSTRACT Purpose In Cushing’s disease (CD) patients, the aim of the present study is to confirm sensitivity of several ACTH and cortisol concentration values in different time points, during corticotropin-releasing hormone (CRH) stimulation test and during CRH stimulation following dexamethasone suppression (DEX-CRH) test. Methods We retrospectively analyzed cortisol and ACTH concentration increment during CRH and DEX-CRH tests in 23 patients with confirmed CD. Cortisol and ACTH concentrations were determined immediately before, 15 min and 30 min after CRH stimulation. We evaluated the sensitivity of different cutoff values including those reported in previous studies, in the diagnosis of CD. Results During DEX–CRH test, 15 min serum cortisol concentration of 1.4 μg/dl (38 nmol/L) had a sensitivity of 90.9%, and serum cortisol concentration ≥1.27 μg/dl (35 nmol/L) had a sensitivity of 100%. For plasma ACTH, sensitivity of 100% was obtained using ACTH ≥3.5pmol/L (16 pg/ml) at 30 min. During CRH test, 35% increase from baseline in ACTH concentration had a sensitivity of 72.7%. Twenty percent increase in cortisol 30 minutes after stimulation yielded a sensitivity of 85.7%. The best sensitivity of ACTH and cortisol increment was obtained 15 min after stimulation, using 19% and 9% increase, respectively (sensitivity of 100% and 92.8%, respectively). Conclusion During CRH and DEX-CRH tests, the study findings agree with the good sensitivity of ACTH and cortisol cutoff values suggested in previous studies; yet, other cutoff values may give a higher diagnostic sensitivity.

The hypothalamic-pituitary-adrenal axis response to ovine corticotropin-releasing-hormone stimulation tests in healthy and hospitalized foals.

The hypothalamic-pituitary-adrenocortical axis (HPAA) response to sepsis can be impaired in critical illness. Corticotropin-releasing hormone (CRH) stimulation test might assess HPAA function in foals. To evaluate plasma cortisol, ACTH, arginine vasopressin (AVP), and endogenous CRH (eCRH) response to different doses of ovine CRH (oCRH). Healthy (n=14) and hospitalized (n=15) foals <7 days of age. In this prospective randomized study, oCRH (0.1, 0.3, and 1μg/kg) was administered intravenously and blood samples were collected before, 15, 30, 60, and 90 minutes after administration of oCRH to determine plasma hormone concentrations. The hormonal response was evaluated as the difference (Delta; μg/dL or pg/mL) or percent change between baseline hormone concentration and each time point after oCRH stimulation. Cortisol concentrations increased from baseline at 15 minutes with 0.1 and 0.3μg/kg and at 30 and 60 minutes from baseline with 1μg/kg oCRH (P < .05) in healthy and hospitalized foals. ACTH concentrations increased from baseline at 15 minutes with 0.1μg/kg and at 30 minutes with 1μg/kg oCRH (P < .05) in hospitalized foals. Delta cortisol 0 - 30, ACTH 0 - 30, and eCRH 0 - 30 was higher for the 1μg/kg compared with 0.1μg/kg oCRH in healthy foals (P < .05). Delta ACTH 0 - 15 and eCRH 0 - 30 was higher for the 1μg/kg compared with the lower doses of oCRH in hospitalized foals (P < .05). Cortisol, ACTH, and eCRH concentrations increased in response to administration of all doses of oCRH. One microgram per kilogram of oCRH appears to be optimal for the assessment of HPAA in healthy and hospitalized foals.

The mechanism of chronic unpredictable mild stress induced high blood pressure in rats: a proteomic and targeted metabolomic analysis.

Chronic stress, a leading factor for high blood pressure (BP) and even hypertension, affects health quality seriously. However, the management is rather difficult in our rapidly developing modern society, and the underlying mechanism that caused hypertension remains incompletely understood. In this study, we established a rat model of high BP induced by chronic unpredictable mild stress (CUMS). The results showed that CUMS increased the BP and heart rate, as well as the concentrations of CORT, NA, and ACTH. Based on tandem mass tag (TMT)-labeled proteomics, 13 proteins changed in RVLM. Then, targeted metabolomics together with real-time qPCR were applied to validate the levels of the biomolecules quantitatively. The related molecules were confirmed to reveal that CUMS has a great role in the upregulation of muscle contraction, synthesis of cAMP and transport of metals, while down-regulating ralaxin signaling. This finding facilitates a better understanding of the mechanism of hypertension induced by chronic stress and could provide an insight into the prevention and treatment of hypertension.

Изучение влияния пептидов продуктов пчеловодства на поведение крыс в условиях хронического стресса

Chronic stress is one of the causes of depression. Various daily factors, accumulating, can lead to the formation of a constant state of stress and anxiety. In this study, we measured the ability of peptides of royal jelly, drone brood, and bee honey up to 5 kDa to reduce anxiety under chronic stress conditions in male Wistar rats. To do this, the animals of the control and experimental groups were subjected to random stress factors for 14 days (2 factors per day), while simultaneously receiving 0.9% NaCl solution (control group) or aqueous solutions of the corresponding peptides intranasally at a concentration of 300 μg/kg of body weight (peptides were obtained by a combination of ultrafiltration, ion exchange chromatography and gel-filtration). After 14 days, the level of anxiety in animals of all groups was measured using such behavioral tests as “Open field”, “Forced swimming” and “Light-dark box”. It was found that in the OF test, animals treated with royal jelly and drone brood peptides show shorter freezing time, higher horizontal and vertical locomotor activity, spend more time on research activities and grooming; in the FST animals of the same groups spent more time actively swimming; in the LDB test animals of the same groups spent more time in the illuminated part of the experimental setup and crossed the border between the dark and light parts more times. The results indicate that rats treated with royal jelly and drone brood peptides have lower levels of stress and anxiety compared to animals that received 0.9% NaCl solution (control group) or a solution of bee honey peptides. After conducting behavioral tests, the animals were withdrawn from the experiment and the concentration of corticosterone and ACTH was determined in the blood serum. It was found that there is no any changes in hormone concentrations between rats from control or experimental groups. Probably, the peptides of drone brood and royal jelly have an anxiolytic effect, without affecting the work of the hypothalamic-pituitary-adrenal axis, but acting through intracerebral molecular targets.

Evaluation of Hormonal Factors in Acne Vulgaris and the Course of Acne Vulgaris Treatment with Contraceptive-Based Therapies in Young Adult Women.

Acne vulgaris is a common chronic inflammatory skin disease, which is considered one of the diseases of civilization due to the significant influence of environmental factors on the severity and frequency of these lesions. The aim of this study was to evaluate the hormonal profile of patients before treatment and to assess selected hormonal parameters after treatment. Our first objective was to examine the correlation between the selected hormonal parameters and the severity of acne before treatment. Our second objective was to evaluate the impact of treatment with three therapies, as measured by the selected hormonal parameters and acne severity. Statistical calculations were performed using the R v.4.1.1 statistical calculation environment (IDE RStudio v. 1.4.1717) with a significance level for the statistical tests set at α = 0.05. The results showed that the women in the pre-treatment (T1) and control (C) groups had significant differences in testosterone, androstendione, FAI, SHBG, prolactin, ACTH, and cortisol concentrations. After treatment, there were still significant differences in testosterone, androstendione, FAI, and SHBG concentrations between the post-treatment (T2) and control groups. We concluded that testosterone, androstendione, and cortisol concentrations correlate with acne severity. Acne in adult women may be an important clinical marker of androgen excess syndrome and cannot be considered a transient symptom of puberty. The mainstay of acne treatment is contraceptive therapy (ethonylestradiol and drospirenone). In this study, we confirmed the effectiveness of three contraceptive-based treatments using hormonal parameters and acne severity.

Status of the Neuroendocrine System in Animals Chronically Exposed to Electromagnetic Fields of 5G Mobile Network Base Stations.

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/cm2) 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.

Plasma Amino Acids in Horses Suffering from Pituitary Pars Intermedia Dysfunction.

Pituitary pars intermedia dysfunction is one of the most common diseases of aged horses and ponies. In Parkinson's disease, which is, similar to PPID, a disease that involves oxidative damage to dopaminergic pathways but with different clinical signs, alterations to the serum amino acid profile have been reported. To examine changes in the plasma amino acid profile in horses with PPID, EDTA plasma of horses that were presented for various reasons that required laboratory examinations of blood anticoagulated with EDTA was collected. With this plasma, the basal ACTH concentration as well as the amino acid profile was determined. Horses were considered PPID patients if the ACTH concentration was ≥ 100 pg/mL, i.e., they would be considered affected at any time. Horses were defined as non-PPID (nPPID) patients if the ACTH concentration was below 30 pg/mL. Horses receiving pergolide with ACTH ≤ 30 pg/mL were allocated to the group PPIDrr (PPID, ACTH in reference range) and horses receiving pergolide with ACTH ≥ 100 pg/mL to the group PPIDarr (PPID, ACTH above reference range). In total, 93 horses were examined, including 88 horses at the clinic and 5 horses at a private practice. Of these, 53 horses fulfilled the inclusion criteria (ACTH ≤ 30 pg/mL or ACTH ≥ 100 pg/mL). A total of 25 horses were diagnosed as nPPID, 20 as PPID, 5 as PPIDrr, and 3 as PPIDarr. Arginine was significantly higher in PPIDrr than in PPID and nPPID, asparagine was significantly higher in PPID, PPIDrr, and PPIDarr than in nPPID, citrulline was significantly higher in PPIDrr than in nPPID and PPID, cysteine was significantly lower in PPIDrr than in PPID, nPPID, and PPIDarr, and glutamine was significantly higher in PPID and PPIDarr than in nPPID. Especially, asparagine, citrulline, and glutamine may be potential diagnostic markers and may offer interesting approaches for research regarding amino supplementation in PPID.

Evaluation of plasma ACTH in the metyrapone test is insufficient for the diagnosis of secondary adrenal insufficiency.

To determine whether a single measurement of ACTH instead of less available in daily practice 11-deoxycortisol assay is sufficient to rule out or confirm secondary adrenal insufficiency (SAI) in the short Metyrapone test. A retrospective analysis of diagnostic tests (Metyrapone and Synacthen tests) performed at our Center between 2016 and 2018 in patients with suspicion of secondary adrenal insufficiency. In 103 patients short metyrapone test was performed with assessment of 11-deoxycortisol and ACTH concentration after Metyrapone administered at midnight. In 89 of them short Synacthen (SST) test was also done (1 or/and 250 mcg 1-24ACTH). ROC curves have been performed to evaluate the diagnostic performance of ACTH level in metyrapone test as the predictor of secondary adrenal insufficiency (SAI) analysing sensitivity and specificity for various possible thresholds proposed in literature. 40 (39%) of examined subjects were diagnosed as SAI, basing on post-Metyrapone 11-deoxycortisol concentration below 70 μg/l. In this group ACTH concentration was 128.1 ng/l (95% CI 96.8-159.4) versus 289.9 ng/l (95% CI 249.1-330.9) in patients with proper adrenal response. There was only a moderate positive correlation between ACTH and 11-deoxycortisol concentrations (r=0.5; p<0.05). The best cut off value of ACTH in relation to 11-deoxycortisol serum concentrations was 147 ng/l - with sensitivity of 73.2% and specificity 83.9%. However, plasma ACTH was>200ng/ml (the highest threshold proposed in literature) in 8 cases (20%) with positive diagnosis of SAI made on the basis of low 11-deoxycortisole and confirmed in short Synacthen test. Our results indicate that for a valuable evaluation of the results of the metyrapone test, the more readily available plasma ACTH assay cannot replace the measurement of 11-deoxycortisol concentrations.

Peripheral glucocorticoid receptor antagonism by relacorilant with modest HPA axis disinhibition.

Glucocorticoid stress hormones are produced in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing's syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing's syndrome.

OR12-2 Inhibition of Basal and ACTH-stimulated Cortisol Secretion in Humans Using an Oral Nonpeptide ACTH Antagonist (CRN04894)

Abstract CRN04894 is a potent orally bioavailable MC2R (adrenal cortex specific ACTH receptor) antagonist (Kb=0.34 nM) that is &amp;gt;1000-fold selective for MC2R over other melanocortin receptor subtypes. In rats receiving continuous administration of ACTH via subcutaneously implanted osmotic pumps, oral administration of CRN04894 over 7 days has previously been shown to result in dose-dependent suppression of basal and ACTH stimulated corticosterone levels. This compound is in clinical development for the treatment of diseases of ACTH excess including congenital adrenal hyperplasia (CAH) and Cushing's Disease. We report initial results from a randomized double-blinded, placebo-controlled single ascending dose study evaluating the safety, pharmacokinetics and pharmacodynamics of CRN04894 in 39 healthy volunteers. After an overnight fast, single doses of CRN04894 were administered at approximately 8 am, 2 hours prior to an IV bolus of ACTH 1-24 (cosyntropin). Serial cortisol over 600 minutes and pharmacokinetics over 168 hours post CRN04894 dose were measured. Two different challenge doses of ACTH 1-24 were studied: 250 μg (supra-pharmacological) and 1 μg (comparable to ACTH concentrations encountered in CAH and Cushing's Disease). CRN04894 was rapidly orally absorbed (median tmax 0.5-1.5 hour), and demonstrated a dose dependent increase in systemic exposure, with an apparent terminal elimination t1/2 of approximately 20 hours. Unstimulated (basal) cortisol measured 2 hours after CRN04894 administration fell in a dose-dependent manner, resulting in reduction near the theoretical maximum with the 80 mg dose cohort (-56.1% [SEM 4.0%, n=12] vs +17.4% in placebo [SEM=18.1%, n=9]). Dose-dependent cortisol suppression following a supra-pharmacological ACTH-stimulated (250 μg) was also observed, with a 41% reduction in the area under the curve (AUC60-600min) post-stimulation at the 80 mg dose. Furthermore, a single dose of 80 mg of CRN04894 reduced the cortisol response (AUC15-120 min) to a disease relevant 1 μg ACTH challenge by 48%, maintaining cortisol concentrations within the normal range seen prior to dosing in a basal unstimulated state. Single doses of CRN04894 were well tolerated with no need for glucocorticoid supplementation. All adverse events (AEs) were considered mild or moderate and there were no serious AEs. The data from this single-dose, proof-of-concept study show that MC2R antagonist CRN04894 was well tolerated after oral delivery in healthy volunteers and demonstrated dose-dependent increases in exposure with lowering of basal and ACTH-stimulated cortisol secretion, including in the presence of disease relevant excess ACTH exposure. Multiple ascending dose evaluations are underway in anticipation of studies in patients with diseases of ACTH excess. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice

ObjectiveThe hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. MethodsPOMC-Cre+/− mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. ResultsIn control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. ConclusionsThese results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Hepatic transcriptomics and metabolomics indicated pathways associated with immune stress of broilers induced by lipopolysaccharide

Broilers with immune stress show decline of growth performance, causing severe economic losses. However, the molecular mechanisms underlying the immune stress still need to be elucidated. One hundred and twenty broiler chicks were randomly assigned to 2 groups with 6 repeats per group, 10 birds per repeat. The model broilers were intraperitoneally injection of 250 µg/kg LPS at 12, 14, 33, and 35 d of age to induce immunological stress. Control group was injected with an equivalent amount of sterile saline. Blood samples from chickens were collected using wing vein puncture at 35 d of age and the serum was obtained for detection of CORT and ACTH. At the end of the experiment, the liver tissues were excised and collected for omics analysis. The results showed that LPS challenge significantly inhibited growth performance, increased relative weight of liver, spleen and decreased relative weight of bursa, as well as enhanced the concentration of serum ACTH and CORT, when compared with the Control. A total of 129 DEGs and a total of 109 differential metabolites were identified between Model and Control group. Transcriptomics profiles revealed that immune stress enhanced the expression of genes related to defense function while declined the expression of genes related to oxidation-reduction process. Metabolomics further suggested that immune stress changed metabolites related to amino acid metabolism, glycerophospholipid metabolism. In addition, integrated analysis suggested that the imbalance of valine, leucine and isoleucine metabolism, glycerophospholipid metabolism, and mTOR signaling pathway played an important role in immune stress of broiler chicks.

Imipramine Can Be Effective on Depressive-Like Behaviors, but Not on Neurotrophic Factor Levels in an Animal Model for Bipolar Disorder Induced by Ouabain.

Despite possible risks of mania switching with the long-term use of antidepressants in patients with bipolar disorder (BD), these drugs may help in depressive episodes. Alterations in neurotrophic factor levels seem to be involved in the pathophysiology of BD. The present study aimed to evaluate the effect of acute treatment of imipramine on behavior and neurotrophic levels in rats submitted to the animal model for BD induced by ouabain. Wistar rats received a single intracerebroventricular (ICV) injection of artificial cerebrospinal fluid or ouabain (10-3M). Following the ICV administration, the rats were treated for 14days with saline (NaCl 0.9%, i.p.), lithium (47.5mg/kg, i.p.), or valproate (200mg/kg, i.p.). On the 13th and 14th days of treatment, the animals received an additional injection of saline or imipramine (10mg/kg, i.p.). Behavior tests were evaluated 7 and 14days after ICV injection. Adrenal gland weight and concentrations of ACTH were evaluated. Levels of neurotrophins BDNF, NGF, NT-3, and GDNF were measured in the frontal cortex and hippocampus by ELISA test. The administration of ouabain induced mania- and depressive-like behavior in the animals 7 and 14days after ICV, respectively. The treatment with lithium and valproate reversed the mania-like behavior. All treatments were able to reverse most of the depressive-like behaviors induced by ouabain. Moreover, ouabain increased HPA-axis parameters in serum and decreased the neurotrophin levels in the frontal cortex and hippocampus. All treatments, except imipramine, reversed these alterations. It can be suggested that acute administration of imipramine alone can be effective on depressive-like symptoms but not on neurotrophic factor alterations present in BD.

Longitudinal assessment of adrenocortical steroid and steroid precursor response to illness in hospitalized foals

Sepsis is a major cause of morbidity and mortality in neonatal foals. Relative adrenal insufficiency (RAI), defined as an inadequate cortisol response to stress, has been associated with sepsis, prematurity, and poor outcome in newborn foals. In addition to cortisol, the adrenal gland synthesizes several biologically important steroids and steroid precursors, including aldosterone, androgens, and progestogens. However, concentration of these hormones during hospitalization and their association with the severity of disease and mortality in critically ill foals have not been completely evaluated. We hypothesized, that in addition to cortisol and aldosterone, concentration of steroid precursors (progestogens and androgens) will be altered in critically ill foals. We also proposed that septic foals will have higher concentrations of steroid precursors than healthy foals, and steroid concentrations will be persistently increased during hospitalization in non-surviving septic and premature foals. Foals <4 days of age were categorized as healthy, septic, sick non-septic, and premature based on physical exam, medical history, and laboratory data. Blood samples were collected on admission (0 h), 24 h, and 72 h after admission. Concentrations of steroids and ACTH were measured by immunoassays. The area under the curve over 72 h (AUC0-72h) of hospitalization was calculated for each hormone. Serum cortisol, aldosterone, progesterone, pregnenolone, dehydroepiandrosterone sulfate (DHEAS), and 17 α-hydroxyprogesterone concentrations were higher in septic and premature foals compared to healthy foals at 0 h and throughout 72 h of hospitalization (P < 0.05). Plasma ACTH concentrations were higher in septic and premature foals on admission compared to healthy controls (P < 0.05). The progesterone (AUC0-72h) cut-off value above which non-survival could be reliably predicted in hospitalized foals was 1,085 ng/mL/h, with 82% sensitivity and 77% specificity. Critically ill neonatal foals had an appropriate response to stress characterized by increased concentrations of cortisol and steroid precursors on admission. A rapid decline in steroid concentration was observed in healthy foals. However, persistently elevated progestogen and androgen concentrations were associated with a lack of improvement in the course of disease and poor outcome.

Approach to the Patient: Diagnosis of Cushing Syndrome.

Cushing syndrome results from supraphysiological exposure to glucocorticoids and is associated with significant morbidity and mortality. The pathogenesis includes administration of corticosteroids (exogenous Cushing syndrome) or autonomous cortisol overproduction, whether or not ACTH-dependent (endogenous Cushing syndrome). An early diagnosis of Cushing syndrome is warranted; however, in clinical practice, it is very challenging partly because of resemblance with other common conditions (ie, pseudo-Cushing syndrome). Initial workup should start with excluding local and systemic corticosteroid use. First-line screening tests including the 1-mg dexamethasone suppression test, 24-hour urinary free cortisol excretion, and late-night salivary cortisol measurement should be performed to screen for endogenous Cushing syndrome. Scalp-hair cortisol/cortisone analysis helps in the assessment of long-term glucocorticoid exposure as well as in detection of transient periods of hypercortisolism as observed in cyclical Cushing syndrome. Interpretation of results can be difficult because of individual patient characteristics and hence requires awareness of test limitations. Once endogenous Cushing syndrome is established, measurement of plasma ACTH concentrations differentiates between ACTH-dependent (80%-85%) or ACTH-independent (15%-20%) causes. Further assessment with different imaging modalities and dynamic biochemical testing including bilateral inferior petrosal sinus sampling helps further pinpoint the cause of Cushing's syndrome. In this issue of "Approach to the patient," the diagnostic workup of Cushing syndrome is discussed with answering the questions when to screen, how to screen, and how to differentiate the different causes. In this respect, the latest developments in biochemical and imaging techniques are discussed as well.