Corticosterone Production Research Articles

6455 The Effect of Gene Silencing on Inhibiting Stress Hormones in Wild Birds Raised Under Stressful Conditions

Abstract Disclosure: S.M. Abdulateef: None. T. Mohammed: None. W. Al-Jugif: None. M.Q. Abed: None. Wild birds are notable for their ease of rearing, tolerance to extreme environmental conditions such as heat and cold, and their resistance to diseases. They are also recognized for their ability to withstand stress. However, their primary challenge is low egg production, stemming from their aggressive nature. This aggression elevates the secretion of stress and fear hormones, significantly impacting reproductive hormones. This study aims to explore the effects of gene silencing on the physiological traits of wild birds, particularly its influence on physiological responses and the reduction of stress hormone levels. Three treatments were employed: T1 (control), T2 (welfare treatment, wherein birds were raised with enhanced well-being), and T3 (using siRNA technology to silence the gene responsible for stress hormone corticosterone production). The silencing of CYP11B1 and CYP11B2 involved obtaining the cDNA of each gene from the NCBI, associated with corticosterone production. The coding sequence from the 5' end at the cap direction was reversed to complement mRNA. Two siRNAs were procured from Macrogene Company (South Korea) in lyophilized form at high concentration, with sequences: CYP11B2 5'-acacctctgcctttgccctgagtgccat-3' and CYP11B1 5'-ctaaagtcaaactcagccccacattcat-3'. These were dissolved in molecular-grade distilled water for a final concentration of 500 pmol/µl. Each hen was injected in the wing in the jugular artery with 125 µl of the siRNA solution at 14 and 18 weeks of age, before the commencement of the egg-laying cycle. Corticosterone levels were measured using the Sunlong Biotech CO., LTD kit. The results showed a significant decrease (p<0.01) in stress hormone levels in the third treatment group (gene silencing treatment), leading to notable physiological responses in the birds compared to the other treatments. This study highlights the impact of gene silencing on the physiological traits of wild birds and its effects on their physiological responses. The findings of this study are significant in the context of avian physiology and poultry farming. By elucidating the impact of stress hormones on egg production and demonstrating the efficacy of gene silencing as a method to enhance reproductive efficiency, this research opens new avenues for improving the productivity and well-being of wild birds in controlled environments. It also underscores the potential of genetic interventions in addressing physiological challenges in wildlife. Presentation: 6/1/2024

12203 FDXR Variants Cause Adrenal Insufficiency And Atypical Sexual Development

Abstract Disclosure: E. Pignatti: None. J. Slone: None. M. Gomez-Cano: None. T.M. Campbell: None. J. Vu: None. K. Sauter: None. A.V. Pandey: None. M. Alonso-Riaño: None. F. Martínez-Azorín: None. D. Neilson: None. N. Longo: None. T. Du Toit: None. C.D. Voegel: None. T. Huang: None. C.E. Flueck: Consulting Fee; Self; Novo Nordisk. Grant Recipient; Self; Novo Nordisk, Sandoz, Pfizer, Inc., Merck. Speaker; Self; Sandoz. Background. Biallelic variants in the mitochondrial flavoprotein ferredoxin—NADP(+)-reductase (FDXR) have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM). FRM patients often experience worsening or demise following stress, but the cause is unclear. Ambiguous genitalia at birth have now been observed in some FRM patients. As FDXR is an essential partner for several steroid enzymes, we explored the impact of FDXR variants on steroid biosynthesis. Methods. To investigate whether the abnormal stress response of many FRM patients is caused by glucocorticoid deficiency, and whether observed ambiguous genitalia is related to androgen excess, we investigated the impact of FDXR variants on adrenal steroidogenesis. Clinical, genetic, biochemical, and functional studies were performed in index patients, steroidogenic cell lines reprogrammed from patient fibroblasts, and a novel disease mouse model. Results. Two paradigmatic female FRM patients carrying novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life had cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. Steroidogenic FDXR-variant cells lines reprogrammed from three patients’ fibroblasts also displayed deficient mineralocorticoid and glucocorticoid production. Likewise, Fdxr-variant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Conclusions. Our comprehensive studies show that FDXR variants in FRM patients may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance. Presentation: 6/1/2024

336 Effects of feeding an immunomodulatory feed additive (NutraGen) on metabolite concentrations and cortisol production in calves following exposure to a combination bovine respiratory disease challenge

Abstract Two 89-d experiments (Exp 1 and Exp 2) evaluated the effects of feeding an immunomodulatory feed additive (NutraGen; NG) in calves following exposure to bovine viral diarrhea virus (BVDV) on d -3 and Mannheimia haemolytica (MH) infection on d 0. For each experiment, steers [body weight (BW) = 251 ± 38.2 kg)] were randomly allocated to 1 of 3 treatments. Treatments included a placebo (CON; Exp 1 n = 5; Exp 2 n = 5), a placebo fed from d -18 to d -3 followed by NG fed from d -3 to d 28 (CHLG; Exp 1 n = 5; Exp 2 n = 6), and NG fed from d -18 to d 28 (PREC; Exp 1 n = 6; Exp 2 n = 5). Data were analyzed using the GLIMMIX procedure of SAS Version 9.4 (SAS Institute, Cary, N.C.). Animal served as the experimental unit and time served as a repeated measure. Treatment, time, and the treatment × time interaction served as fixed effects. Block was included as a random effect. Multiple treatment × time interactions and time effects occurred for serum glucose, lactate, blood urea nitrogen, and non-esterified fatty acid concentrations (P ≤ 0.003) in both experiments. There was a general trend for serum glucose to decrease over time post-challenge, suggesting increased cellular utilization of glucose in response to the challenge. In Exp 1, treatment effects occurred for serum glucose (P ≤ 0.001) and serum lactate (P ≤ 0.01), where calves assigned to the CHLG treatment displayed the greatest concentrations of the metabolites. Blood urea nitrogen concentrations peaked at h 48 in Exp 1 and h 6 in Exp 2. There were no treatment effects or treatment × time interactions for serum cortisol, hair cortisol, or fecal corticosterone concentrations in either experiment (P ≥ 0.17). There were, however, multiple time effects for serum cortisol, hair cortisol, and fecal corticosterone concentrations in both experiments (P ≤ 0.02), indicating that the challenge directly impacted cortisol and corticosterone production. Serum cortisol steadily increased from h 96 to h 168 in both experiments. Trends in cortisol and corticosterone production were similar in both experiments; however, peak concentration times for these variables occurred later in Exp 2. These data suggest that feeding NG to calves may alter glucose availability and nutrient utilization in response to infection resulting from a bovine respiratory disease challenge.

Co-culture of vascular endothelial cells enhances corticosterone production in steroid hormone-producing cells generated from adipose-derived mesenchymal stromal cells

Cell therapy for adrenocortical insufficiency can potentially provide steroid replacement in response to physiological stimuli. Previously, we reported that adipose tissue-derived stromal cells (ADSCs) are transformed into steroid-producing cells by overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1). The steroidogenic cells are characterized by the production of both adrenal and gonadal steroids. Cytotherapy for adrenocortical insufficiency requires cells with more adrenocortical characteristics. Considering the highly developed vascular network within the adrenal cortex, all adrenocortical cells are adjacent to and interact with vascular endothelial cells (VECs). In this study, NR5A1-induced steroidogenic cells derived from mouse ADSCs (NR5A1-ADSCs) were co-cultured with mouse VECs. Testosterone secretion in NR5A1-ADSCs was not altered; however, corticosterone secretion significantly increased while levels of steroidogenic enzymes significantly increased in the corticosterone synthesis pathway. Co-culture with lymphatic endothelial cells (LECs) or ADSCs, or transwell culture with NR5A1-ADSCs and VECs did not alter corticosterone production. VECs expressed higher levels of collagen and laminin than LECs. Culture in type-IV collagen and laminin-coated dishes increased corticosterone secretion in NR5A1-ADSCs. These results suggest that VECs may characterize ADSC-derived steroidogenic cells into a more corticosterone-producing phenotype, and VECs may be useful for generating adrenal steroidogenic cells from stem cells.

FDXR variants cause adrenal insufficiency and atypical sexual development.

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

Circadian Corticosterone Profile in Laying Hens (Gallus gallus domesticus).

Measurement of blood corticosterone concentrations has been established as an indicator for assessment of acute distress. Therefore, knowledge on physiological fluctuations is required, but previous studies allow little conclusion on daily fluctuations in domestic chickens (Gallus gallus domesticus). To verify the presence of a circadian corticosterone rhythm in socialized chickens, blood samples were taken at four-hour intervals from 12 laying hens kept in groups of four over three days, each. Prior to experiments, hens were adapted to repeated handling for stress reduction. Corticosterone concentration was determined using radioimmunoassay. Blood sampling time and duration were recorded, and audio and video recordings were analyzed to assess the impact of behavior on corticosterone concentrations. Despite individual fluctuations, most hens showed a circadian course with two peaks per day. Statistics revealed a significant peak during the day (between 12:00 p.m. and 04:00 p.m.) and a tendency for a second peak at night (12:00 a.m.). The daily corticosterone peak was not explained by daytime social stress and needs to be seen as an endophenotype. The role of nightly corticosterone production has to be investigated in further studies. There might be a relation between corticosterone and reproduction since the only hen not showing peaks was not laying eggs.

Adrenal Abcg1 Controls Cholesterol Flux and Steroidogenesis.

Cholesterol is the precursor of all steroids, but how cholesterol flux is controlled in steroidogenic tissues is poorly understood. The cholesterol exporter ABCG1 is an essential component of the reverse cholesterol pathway and its global inactivation results in neutral lipid redistribution to tissue macrophages. The function of ABCG1 in steroidogenic tissues, however, has not been explored. To model this, we inactivated Abcg1 in the mouse adrenal cortex, which led to an adrenal-specific increase in transcripts involved in cholesterol uptake and de novo synthesis. Abcg1 inactivation did not affect adrenal cholesterol content, zonation, or serum lipid profile. Instead, we observed a moderate increase in corticosterone production that was not recapitulated by the inactivation of the functionally similar cholesterol exporter Abca1. Altogether, our data imply that Abcg1 controls cholesterol uptake and biosynthesis and regulates glucocorticoid production in the adrenal cortex, introducing the possibility that ABCG1 variants may account for physiological or subclinical variation in stress response.

Pseudohypoadrenalism, a subclinical cortisol metabolism disorder in hyperuricemia.

Hyperuricemia is a known risk factor of lipid metabolism disorder. However, the mechanisms have not been fully understood. The serum samples from hyperuricemia subjects were used to analyze the correlation between serum uric acid and clinical characteristics. Hyperuricemia mice induced by potassium oxonate (PO) and adenine were used to explore glucocorticoid metabolism. In hyperuricemia patients, the levels of serum uric acid were positively correlated with the levels of γ-glutamyltransferase, associated with a cortisol metabolism disorder. In hyperuricemia state, the adrenal glands failed to respond to adrenocorticotropic hormone properly, leading to low cortisol, but not corticosterone production, and decreased mRNA levels of aldosterone synthase, 11β-hydroxylase, and 3β-hydroxysteroid dehydrogenase 1, three key enzymes for cortisol synthesis. The expression of both hepatic 5α-reductase and renal 11β-hydroxysteroid dehydrogenase 2 was significantly reduced, which led to low cortisol clearance. We denominated this cortisol metabolism disorder in hyperuricemia as pseudohypoadrenalism (PHAL). PHAL increased exposure to the bioavailable cortisol in the liver, leading to local amplification of the biological action of corticosteroids. Unregulated biosynthesis pathway of bile acid expanded bile acid pool, and further aggravated cholestatic liver injury.

Limonin ameliorates cisplatin-induced acute liver injury by inhibiting 11β-hydroxysteroid dehydrogenase type 1

BackgroundAcute liver injury (ALI) is a common side effect of cisplatin treatment in the clinic and can lead to liver failure if not treated promptly. Previous studies have revealed that Limonin, a critical bioactive substance in citrus fruits, can protect multiple organs from various medical conditions. However, whether Limonin could ameliorate cisplatin-induced ALI remains unclear. MethodsIn vivo and in vitro models were induced by cisplatin in the present study. Non-targeted metabolomics was employed to analyze the metabolic changes in the liver after ALI. In addition, molecular docking was utilized to predict the potential targets of Limonin. ResultsLimonin attenuated hepatic histopathological injury by reducing hepatocyte apoptosis, lipid peroxidation, and inflammation in cisplatin-challenged mice. Employing metabolomics, we revealed that Limonin mediated the balance of various disturbed metabolic pathways in the liver after cisplatin-induced ALI. Integrating public data mining, molecular docking studies, and in vitro experiments demonstrated that Limonin suppressed the expression and activity of its direct target, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), in the liver, thus reducing the production of corticosterone (CORT), a key metabolite promoted hepatocyte apoptosis. ConclusionsLimonin improves the liver metabolic microenvironment by inhibiting 11β-HSD1 to protect against cisplatin-induced ALI.

OR27-05 Two-pronged Mediation Of Adrenal Steroidogenesis By Oxytocin

Abstract Disclosure: U. Cheliadinova: None. G. Pevnev: None. V. Ryu: None. T. Frolinger: None. S.L. Sims: None. M. Ofer: None. F. Korkmaz: None. O. Barak: None. J. Gimenez Roig: None. F. Sultana: None. N. Kramskiy: None. S. Wizman: None. M. Orloff: None. T. Yuen: None. D. Lizneva: None. M. Zaidi: None. A. Gumerova: None. Stress–stimulated glucocorticoid release affects diverse biological processes, including pregnancy and childbirth. The controversy surrounding effects of OXT on stress prompted us to investigate its actions on adrenal cortical function in mice. RNAscope and immunohistochemistry provided unequivocal evidence for abundant OXT receptor (OXTR) expression in the adrenal cortex, predominantly in zona fasciculata and zona reticularis. Female mice displayed higher adrenal cortical Oxtr expression, more than a range of other tissues under study. Oxtr expression was also detected in the adenohypophysis solely in female mice, while both male and female mice expressed Oxtr in the pars intermedia of the hypophysis. Aging impressively attenuated Oxtr expression in female mice in both the adrenal gland and adenohypophysis. For loss–of–function studies, we generated tissue–specific Star-CreERT2/Oxtrfl/fl mice in which the Oxtr was deleted from steroidogenic tissues, namely adrenals and ovaries. Deletion was confirmed by RNAscope and immunohistochemistry. We found no difference in serum corticosterone between tamoxifen–induced and uninduced mice; however, the elevation of serum corticosterone upon ACTH stimulation or after four weeks of stress was attenuated in tamoxifen–induced mice. Bulk RNAseq of adrenals from tamoxifen–induced mice revealed the downregulation of key steroidogenic genes, namely Cyp17a1, Hsd3b3, Hsd17b2, Cyp3a41b, and Serpina6 (a corticosteroid–binding globulin). In gain–of–function studies, a single OXT injection led to a rise of serum ACTH in tamoxifen–induced mice, which was greater in magnitude than uninduced controls. However, after five days of OXT injections, corticosterone levels became elevated, whereas ACTH levels declined to baseline. This suggests that OXT acts directly on OXTRs on corticotropes to initiate a stress–like response, with a negative feedback loop that inhibits further ACTH secretion. Of note is that male mice did not display any difference between tamoxifen–induced and uninduced groups, consistent with their low hypophyseal Oxtr expression. In all, we describe a novel gender–specific two–pronged circuit through which OXT may precisely regulates the production of corticosterone from the adrenal cortex. In the first arm, OXT directly stimulates ACTH secretion through adenohypophysis OXTRs. In the second arm, OXT prevents excessive stress–induced adrenal stimulation by downregulating steroidogenic genes. Our discovery lays the foundation for the emergence of new physiology that may relate to a fundamental role for oxytocin in the stress response during procreation, notably, when levels are elevated during pregnancy and lactation. Presentation: Saturday, June 17, 2023

Hepatic-Specific FGF21 Knockout Abrogates Ovariectomy-Induced Obesity by Reversing Corticosterone Production.

Increased glucocorticoid (GC) levels act as a master contributor to central obesity in estrogen-depleted females; however, what factors cause their increased GC production is unclear. Given (1) liver fibroblast growth factor 21 (FGF21) and GCs regulate each other's production in a feed-forward loop, and (2) circulating FGF21 and GCs are parallelly increased in menopausal women and ovariectomized mice, we thus hypothesized that elevation of hepatic FGF21 secretion causes increased GGs production in estrogen-depleted females. Using the ovariectomized mice as a model for menopausal women, we found that ovariectomy (OVX) increased circulating corticosterone levels, which in turn increased visceral adipose Hsd11b1 expression, thus causing visceral obesity in females. In contrast, liver-specific FGF21 knockout (FGF21 LKO) completely reversed OVX-induced high GCs and high visceral adipose Hsd11b1 expression, thus abrogating OVX-induced obesity in females. Even though FGF21 LKO failed to rescue OVX-induced dyslipidemia, hepatic steatosis, and insulin resistance. What's worse, FGF21 LKO even further exacerbated whole-body glucose metabolic dysfunction as evidenced by more impaired glucose and pyruvate tolerance and worsened insulin resistance. Mechanically, we found that FGF21 LKO reduced circulating insulin levels, thus causing the dissociation between decreased central obesity and the improvement of obesity-related metabolic syndromes in OVX mice. Collectively, our results suggest that liver FGF21 plays an essential role in mediating OVX-induced central obesity by promoting GC production. However, lack of liver FGF21 signaling reduces insulin production and in turn causes the dissociation between decreased central obesity and the improvement of obesity-related metabolic syndromes, highlighting a detrimental role for hepatic FGF21 signals in mediating the development of central obesity but a beneficial role in preventing metabolic abnormality from further exacerbation in estrogen-depleted females.

ACTH and prolactin synergistically and selectively regulate CYP17 expression and adrenal androgen production in human foetal adrenal organ cultures.

The essential role of ACTH on the growth and function of the human foetal adrenal (HFA) has long been recognized. In addition, many studies have suggested a role of the pituitary hormone prolactin (PRL) in the regulation of the HFA, but the effects of this hormone on steroidogenesis and gene expression are still unknown. Our objective was to investigate the effect of ACTH and PRL on the steroidogenic capacities of the HFA. In vitro/ex vivo experimental study. We used a hanging drop in vitro organ culture system. First trimester HFA samples were cultured for 14 days in basal conditions or treated with ACTH, PRL, or a combination of the 2 (3 to 11 replicates depending on the experiment). Steroids were measured by liquid chromatography/tandem mass spectrometry or immunoassay, gene expression by RT-qPCR, and protein expression by immunoblot. ACTH significantly increased corticosterone, cortisol, and cortisone production, both by itself and when used together with PRL. PRL stimulation by itself had no effect. Combined stimulation with ACTH + PRL synergistically and selectively increased adrenal androgen (DHEAS and Δ4-androstenedione) production and CYP17A1 expression in the HFA, while treatment with each single hormone had no significant effect on those steroids. These results have important implications for our understanding of the hormonal cues regulating adrenal steroidogenesis in the HFA during the first trimester in physiological and pathological conditions and warrant further studies to characterize the molecular mechanisms of converging ACTH and PRL signalling to regulate CYP17A1 expression.

Grape Polyphenols May Prevent High-Fat Diet-Induced Dampening of the Hypothalamic-Pituitary-Adrenal Axis in Male Mice.

Chronic high-fat diet (HFD) consumption causes obesity associated with retention of bile acids (BAs) that suppress important regulatory axes, such as the hypothalamic-pituitary-adrenal axis (HPAA). HFD impairs nutrient sensing and energy balance due to a dampening of the HPAA and reduced production and peripheral metabolism of corticosterone (CORT). We assessed whether proanthocyanidin-rich grape polyphenol (GP) extract can prevent HFD-induced energy imbalance and HPAA dysregulation. Male C57BL6/J mice were fed HFD or HFD supplemented with 0.5% w/w GPs (HFD-GP) for 17 weeks. GP supplementation reduced body weight gain and liver fat while increasing circadian rhythms of energy expenditure and HPAA-regulating hormones, CORT, leptin, and PYY. GP-induced improvements were accompanied by reduced mRNA levels of Il6, Il1b, and Tnfa in ileal or hepatic tissues and lower cecal abundance of Firmicutes, including known BA metabolizers. GP-supplemented mice had lower concentrations of circulating BAs, including hydrophobic and HPAA-inhibiting BAs, but higher cecal levels of taurine-conjugated BAs antagonistic to farnesoid X receptor (FXR). Compared with HFD-fed mice, GP-supplemented mice had increased mRNA levels of hepatic Cyp7a1 and Cyp27a1, suggesting reduced FXR activation and more BA synthesis. GP-supplemented mice also had reduced hepatic Abcc3 and ileal Ibabp and Ostβ, indicative of less BA transfer into enterocytes and circulation. Relative to HFD-fed mice, CORT and BA metabolizing enzymes (Akr1d1 and Srd5a1) were increased, and Hsd11b1 was decreased in GP supplemented mice. GPs may attenuate HFD-induced weight gain by improving hormonal control of the HPAA and inducing a BA profile with less cytotoxicity and HPAA inhibition, but greater FXR antagonism.

DIETS THAT HAVE POTENTIAL TO STIMULATE THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS IN SPRAGUE DAWLEY RATS

The pituitary gland is the master gland which regulates the body hormones. From the corticotrophs of the anterior pituitary gland, adrenocorticotrophic hormone (ACTH) is secreted and influences corticosterone production in rodents via the hypothalamic–pituitary–adrenal axis (HPA axis). ACTH and corticosterone are frequently used as a hormonal marker of stress level. Disturbance in ACTH and corticosterone were observed in many diseases especially Addison’s disease, Cushing’s syndrome, and metabolic syndrome. Our objective is to observe which diet produces the most and least stress to the Sprague Dawley rat’s physiology. Method: Thirty-five Sprague Dawley rats were grouped into five groups (n=7) and were given five different types of diets (control, high-fat, high-protein, high-sugar, and high-starch) with tap water supplied as drinking water ad libitum. By the end of eight weeks, the rat’s blood was collected, and serum separated. The individual components of ACTH and corticosterone from the blood was extracted, purified, identified, and quantified using the High-Performance Liquid Chromatography (HPLC) with photodiode array (PDA) analysis method. Result: The analytical studies revealed a high -sugar diet registered highest ACTH blood level. It was also revealed that the high-fat and high-sugar diet group presented with the highest peak for corticosterone. Conclusion: Consumption of high-fat and high-sugar diet for eight weeks is suggested to induce physiologic and metabolic stress in Sprague Dawley rats as evidenced by the HPA activation.

Pathogen odors elicit distinct neurosteroid and behavioral responses in Rainbow trout (Oncorhynchus mykiss)

Rainbow trout can smell virus and bacteria cues that in turn activate an immune response in the brain in absence of a systemic response (i.e. the immune response is activated by the nervous system instead of the pathogen inside tissues or bloodstream). However, little is known if pathogen odors can elicit different physiological responses (i.e. behavioral and immune) and how are pathogen odors integrated into different responses in the brain. We hypothesize that pathogen odors not only activate immune responses but also induce avoidance behavior. Moreover, we hypothesized that pathogen olfactory responses are modulated by neurosteroids at different times in different parts of the brain. We used a two-choice maze test to determine whether inactivated Yersinia ruckeri elicits aversive behavior. Furthermore, to check if behavioral and immune responses are modulated by neurosteroids, we performed a second experiment where we nasally administered inactivated Y. ruckeri to rainbow trout. After that, the nose, olfactory bulb, cerebellum, and optic tectum were collected at four different time points (15 min, 4 hours, 24 hours, and 7 days) and samples were extracted to measure gene expression of several steroidogenic enzymes ( cyp19a1, 3βHSD, 11HSD, and 17HSD) and fish production of neurosteroids (e.g. cortisol, estradiol, and progesterone). We found that rainbow trout actively avoids pathogen odors. Moreover, we found a different expression of neuro steroidogenic enzymes at different brain areas and times post-exposure. Fish exposed to pathogen odor express a significant increase (ANOVA 2 factor, P<0.05) of cyp19a1 (involved in estradiol synthesis) as soon as 15 min in the nose and olfactory bulb, and 4 hours post-exposure in the cerebellum and optic tectum. This gene expression was parallel to a significant increase of estradiol in the nose, bulb, and rest of the brain between 15 min and 4 hours. Moreover, there was a significant increase of 11HSD and 17HSD (related to the production of testosterone, cortisol, and corticosterone) 4 hours after exposure in the nose and cerebellum but a significant decrease in the olfactory bulb and optic tectum. These gene expression patterns were parallel o an increase of cortisol in the nose and olfactory bulb. Finally, we found a significant increase of 3βHSD (involved in the first steps of steroidogenesis) after 24 hours in the nose and olfactory bulb, although we didn’t find an increase in steroid production in our preliminary assays. Our results showed that exposure to pathogen odors activates different neuro steroidogenic pathways at different times after exposure, and at different areas of the brain. These findings pinpoint the use of the sense of smell by fish to control behavioral and immune responses to pathogens, with neurosteroids playing a key role in modulating and integrating these olfactory responses. This material is based upon work supported by the National Science Foundation under Grant No. 1755348 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early life stress in mice leads to impaired colonic corticosterone production and prolonged inflammation following induction of colitis

Early life stress (ELS) is an environmental trigger linked to increased risk of inflammatory bowel disease (IBD). Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to, and/or severity of, intestinal inflammation. We hypothesized that ELS alters the delicate balance of typical homeostatic mediators in the intestinal microenvironment, predisposing ELS-exposed individuals to IBD onset or worsened disease upon a pro-inflammatory triggering event within the gut. We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4-8 hours beginning on postnatal day 2, then weaned early on postnatal day 17. Normally reared (NR) control mice were left undisturbed with the dams until standard weaning on postnatal day 21. In the second model, dams were fed the synthetic glucocorticoid – dexamethasone, or vehicle ad libitum in drinking water (3 μg/mL) on post-partum days 1-14. Colitis was induced in 4-week-old mice via intraperitoneal injection of IL-10 receptor blocking antibody (100 μg) every 5 days for 15 days. Five or 15 days later, histological colitis scores and colonic transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. ELS-exposed mice displayed altered mucosa-associated immune cell balances, with an initial deficit in anti-inflammatory IL-10-producing CD4 T cells at 4-wks of age (n≥11, p<0.05), followed by an increase in IFNγ-producing CD4 T cells at 8-wks of age (n≥7, p<0.05). ELS-exposed mice also displayed reduced levels of the glucocorticoid, corticosterone in the plasma (n≥10, p<0.01) as well as locally within the colon (n≥7, p<0.05). Upon colitis induction, NR control animals showed improvements in indices of inflammation following cessation of IL-10 receptor blockade, whereas ELS-exposed animals maintained high levels of intestinal Tnf (n≥7, p<0.01) and histological signs of colitis (n≥7, p<0.05). In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis ( Cyp11a1; n≥5, p<0.05) and responsiveness ( Nr3c1, Nr5a2; n≥5, p<0.01). Finally, ex-vivo TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis, relative to NR control mice (n≥4, p<0.05). Our study identifies impaired local colonic glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts. Crohn's and Colitis Foundation Research Initiatives Award This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Mycobacterium avium infection differentially affects the cytokine expression profile of three mouse strains, but has no effect on behavior

One of the most remarkable findings in the immunology and neuroscience fields was the discovery of the bidirectional interaction between the immune and the central nervous systems. This interplay is tightly regulated to maintain homeostasis in physiological conditions. Disruption in this interplay has been suggested to be associated with several neuropsychiatric disorders. Most studies addressing the impact of an immune system disruption on behavioral alterations focus on acute pro-inflammatory responses. However, chronic infections are highly prevalent and associated with an altered cytokine milieu that persists over time. Studies addressing the potential effect of mycobacterial infections on mood behavior originated discordant results and this relationship needs to be further addressed. To increase our understanding on the effect of chronic infections on the central nervous system, we evaluated the role of Mycobacterium avium infection. A model of peripheral chronic infection with M. avium in female from three mouse strains (Balb/c, C57BL/6, and CD-1) was used. The effect of the infection was evaluated in the cytokine expression profile (spleen and hippocampus), hippocampal cell proliferation, neuronal plasticity, serum corticosterone production and mood behavior. The results show that M. avium peripheral chronic infection induces alterations not just in the peripheral immune system but also in the central nervous system, namely in the hippocampus. Interestingly, the cytokine expression profile alterations vary between mouse strains, and are not accompanied by hippocampal cell proliferation or neuronal plasticity changes. Accordingly, no differences were observed in locomotor, anxious and depressive-like behaviors, in any of the mouse strains used. We conclude that the M. avium 2447 infection-induced alterations in the cytokine expression profile, both in the periphery and the hippocampus, are insufficient to alter hippocampal plasticity and behavior.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Cortisol/glucocorticoid receptor: a critical mediator of the ovulatory process and luteinization in human periovulatory follicles.

Do cortisol/glucocorticoid receptors play an active role in the human ovary during ovulation and early luteinization? The ovulatory hCG stimulation-induced glucocorticoid receptor signaling plays a crucial role in regulating steroidogenesis and ovulatory cascade in human periovulatory follicles. Previous studies reported an increase in cortisol levels in the human follicular fluid after the LH surge or ovulatory hCG administration. However, little is known about the role of cortisol/glucocorticoid receptors in the ovulatory process and luteinization in humans. This study was an experimental prospective clinical and laboratory-based study. An in vivo experimental study was accomplished utilizing the dominant ovarian follicles from 38 premenopausal women undergoing laparoscopic sterilization. An in vitro experimental study was completed using the primary human granulosa/lutein cells (hGLC) from 26 premenopausal women undergoing IVF. This study was conducted in a private fertility clinic and academic medical centers. Dominant ovarian follicles were collected before the LH surge and at defined times after hCG administration from women undergoing laparoscopic sterilization. Primary hGLC were collected from women undergoing IVF. hGLC were treated without or with hCG in the absence or presence of RU486 (20 µM; dual antagonist for progesterone receptor and glucocorticoid receptor) or CORT125281 (50 µM; selective glucocorticoid receptor antagonist) for 12 or 36 h. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and ovulatory cascade was studied with RT-quantitative PCR and western blotting. The production of cortisol, corticosterone, and progesterone was assessed by hormone assay kits. hCG administration upregulated the expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), nuclear receptor subfamily 3 group C member 1 (NR3C1), FKBP prolyl isomerase 5 (FKBP5), and FKBP prolyl isomerase 4 (FKBP4) in human ovulatory follicles and in hGLC (P < 0.05). RU486 and CORT125281 reduced hCG-induced increases in progesterone and cortisol production in hGLC. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and the key ovulatory process was reduced by RU486 and/or CORT125281 in hGLC. N/A. The role of cortisol/glucocorticoid receptors demonstrated using the hGLC model may not fully reflect their physiological roles in vivo. Successful ovulation and luteinization are essential for female fertility. Women with dysregulated cortisol levels often suffer from anovulatory infertility. Deciphering the functional role of glucocorticoid receptor signaling in human periovulatory follicles enhances our knowledge of basic ovarian physiology and may provide therapeutic insights into treating infertility in women. This study was supported by P01HD71875 (to M.J., T.E.C., and M.B.) and R01HD096077 (to M.J.) from the Foundation for the National Institutes of Health and the BTPSRF of the University of Kentucky Markey Cancer Center (P30CA177558). The authors report no competing interests. N/A.

Early Life Stress in Mice Leads to Impaired Colonic Corticosterone Production and Prolonged Inflammation Following Induction of Colitis.

Early life stress (ELS) is an environmental trigger believed to promote increased risk of IBD. Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to and/or severity of gut inflammation. We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4 to 8 hours starting on postnatal day 2 and then weaned early on postnatal day 17. Control mice were left undisturbed with the dams until weaning on postnatal day 21. In the second model, dams were fed dexamethasone or vehicle ad libitum in drinking water on postpartum days 1 to 14. Plasma and colonic corticosterone were measured in juvenile and adult mice. Colitis was induced in 4-week-old mice via intraperitoneal injection of interleukin (IL)-10 receptor blocking antibody every 5 days for 15 days. Five or 15 days later, colitis scores and transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. Mice exposed to ELS displayed reduced plasma and colonic corticosterone. Control animals showed improvements in indices of inflammation following cessation of interleukin-10 receptor blockade, whereas ELS-exposed animals maintained high levels of Tnf and histological signs of colitis. In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis and responsiveness. Finally, TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis. Our study identifies impaired local glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts.