Glucocorticoid Levels Research Articles

11β-Hydroxysteroid dehydrogenase and the brain: Not (yet) lost in translation.

11-beta-hydroxysteroid dehydrogenases (11β-HSDs) catalyse the conversion of active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inert 11-keto forms (cortisone, 11-dehydrocorticosterone). They were first reported in the body and brain 70 years ago, but only recently have they become of interest. 11β-HSD2 is a dehydrogenase, potently inactivating glucocorticoids. In the kidney, 11β-HSD2 generates the aldosterone-specificity of intrinsically non-selective mineralocorticoid receptors. 11β-HSD2 also protects the developing foetal brain and body from premature glucocorticoid exposure, which otherwise engenders the programming of neuropsychiatric and cardio-metabolic disease risks. In the adult CNS, 11β-HSD2 is confined to a part of the brain stem where it generates aldosterone-specific central control of salt appetite and perhaps blood pressure. 11β-HSD1 is a reductase, amplifying active glucocorticoid levels within brain cells, notably in the cortex, hippocampus and amygdala, paralleling its metabolic functions in peripheral tissues. 11β-HSD1 is elevated in the ageing rodent and, less certainly, human forebrain. Transgenic models show this rise contributes to age-related cognitive decline, at least in mice. 11β-HSD1 inhibition robustly improves memory in healthy and pathological ageing rodent models and is showing initial promising results in phase II studies of healthy elderly people. Larger trials are needed to confirm and clarify the magnitude of effect and define target populations. The next decade will be crucial in determining how this tale ends - in new treatments or disappointment.

Long-term glucocorticoids in relation to the metabolic syndrome and cardiovascular disease: A systematic review and meta-analysis.

The striking link of Cushing's syndrome with the metabolic syndrome (MetS) and cardiovascular disease (CVD) suggests that long-term exposure to extremely high cortisol levels catalyzes cardiometabolic deterioration. However, it remained unclear whether the findings from the extreme glucocorticoid overabundance observed in Cushing's syndrome could be translated into more subtle variations in long-term glucocorticoid levels among the general population, for example, due to chronic stress. Here, we performed a systematic review (PROSPERO: CRD42023425541) of evidence regarding the role of subtle variations in long-term biological stress, measured as levels of scalp hair cortisol (HairF) and cortisone (HairE), in the context of MetS and CVD in adults. We also performed a meta-analysis on the cross-sectional difference in HairF levels between individuals with versus without CVD. Seven studies were included regarding MetS, sixteen regarding CVD, and one regarding both. Most articles indicated a strong, consistent cross-sectional association of higher HairF and HairE levels with CVD, which was confirmed by our meta-analysis for HairF (eight studies, SMD=0.48, 95% confidence intervals [CIs]: 0.16-0.79, p=0.0095). Moreover, these relationships appear largely independent of standard risk factors. Age seems relevant as the effect seems stronger in younger individuals. Results regarding the associations of HairF and HairE with MetS were inconsistent. Altogether, long-term biological stress, measured as HairF and HairE, is associated with the presence of CVD, and less consistently with MetS. Prospective studies need to evaluate the directionality of this relationship and determine whether HairF and HairE can be used in addition to standard risk factors in predicting future cardiometabolic deterioration.

Intrauterine food restriction impairs the lipogenesis process in the mesenteric adipocytes from low-birth-weight rats into adulthood.

Intrauterine food restriction (IFR) during pregnancy is associated with low birth weight (LBW) and obesity in adulthood. It is known that white adipose tissue (WAT) plays critical metabolic and endocrine functions; however, this tissue's behavior before weight gain and obesity into adulthood is poorly studied. Thus, we evaluated the repercussions of IFR on the lipogenesis and lipolysis processes in the offspring and described the effects on WAT inflammatory cytokine production and secretion. We induced IFR by providing gestating rats with 50% of the necessary chow daily amount during all gestational periods. After birth, we monitored the offspring for 12 weeks. The capacity of isolated fat cells from mesenteric white adipose tissue (meWAT) to perform lipogenesis (14C-labeled glucose incorporation into lipids) and lipolysis (with or without isoproterenol) was assessed. The expression levels of genes linked to these processes were measured by real-time PCR. In parallel, Multiplex assays were conducted to analyze pro-inflammatory markers, such as IL-1, IL-6, and TNF-α, in the meWAT. Twelve-week-old LBW rats presented elevated serum triacylglycerol (TAG) content and attenuated lipogenesis and lipolysis compared to control animals. Inflammatory cytokine levels were increased in the meWAT of LBW rats, evidenced by augmented secretion by adipocytes and upregulated gene and protein expression by the tissue. However, there were no significant alterations in the serum cytokines content from the LBW group. Additionally, liver weight, TAG content in the hepatocytes and serum glucocorticoid levels were increased in the LBW group. The results demonstrate that IFR throughout pregnancy yields LBW offspring characterized by inhibited lipogenesis and lipolysis and reduced meWAT lipid storage at 12 weeks. The increased serum TAG content may contribute to the augmented synthesis and secretion of pro-inflammatory markers detected in the LBW group.

Meta-analysis reveals glucocorticoid levels reflect variation in metabolic rate, not ‘stress’

Glucocorticoid (GC) variation has long been thought to reflect variation in organismal ‘stress,’ but associations between GCs and Darwinian fitness components are diverse in magnitude, direction, and highly context-dependent. This paradox reveals our poor understanding of the causes of GC variation, contrasting with the detailed knowledge of the functional consequences of GC variation. Amongst an array of effects in many physiological systems, GCs orchestrate energy availability to anticipate and recover from predictable and unpredictable environmental fluctuations and challenges. Although this is mechanistically well-known, the extent to which GC levels are quantitatively explained by energy metabolism is unresolved. We investigated this association through meta-analysis, selecting studies of endotherms in which (1) an experiment was performed that affected metabolic rate and (2) metabolic rate and GC levels were measured simultaneously. We found that an increase in metabolic rate was associated with an increase in GC levels in 20 out of 21 studies (32 out of 35 effect sizes). More importantly, there was a strong positive correlation between the increases in metabolic rate and GCs (p=0.003). This pattern was similar in birds and mammals, and independent of the nature of the experimental treatment. We conclude that metabolic rate is a major driver of GC variation within individuals. Stressors often affect metabolic rate, leading us to question whether GC levels provide information on ‘stress’ beyond the stressor’s effect on metabolic rate.

Meta-analysis reveals glucocorticoid levels reflect variation in metabolic rate, not 'stress'.

Glucocorticoid (GC) variation has long been thought to reflect variation in organismal 'stress,' but associations between GCs and Darwinian fitness components are diverse in magnitude, direction, and highly context-dependent. This paradox reveals our poor understanding of the causes of GC variation, contrasting with the detailed knowledge of the functional consequences of GC variation. Amongst an array of effects in many physiological systems, GCs orchestrate energy availability to anticipate and recover from predictable and unpredictable environmental fluctuations and challenges. Although this is mechanistically well-known, the extent to which GC levels are quantitatively explained by energy metabolism is unresolved. We investigated this association through meta-analysis, selecting studies of endotherms in which (1) an experiment was performed that affected metabolic rate and (2) metabolic rate and GC levels were measured simultaneously. We found that an increase in metabolic rate was associated with an increase in GC levels in 20 out of 21 studies (32 out of 35 effect sizes). More importantly, there was a strong positive correlation between the increases in metabolic rate and GCs (p=0.003). This pattern was similar in birds and mammals, and independent of the nature of the experimental treatment. We conclude that metabolic rate is a major driver of GC variation within individuals. Stressors often affect metabolic rate, leading us to question whether GC levels provide information on 'stress' beyond the stressor's effect on metabolic rate.

Thymic atrophy induced by Plasmodium berghei ANKA and Plasmodium yoelii 17XL infection

The thymus is the anatomical site where T cells undergo a complex process of differentiation, proliferation, selection, and elimination of autorreactive cells which involves molecular signals in different intrathymic environment. However, the immunological functions of the thymus can be compromised upon exposure to different infections, affecting thymocyte populations. In this work, we investigated the impact of malaria parasites on the thymus by using C57BL/6 mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL; these lethal infection models represent the most severe complications, cerebral malaria, and anemia respectively. Data showed a reduction in the thymic weight and cellularity involving different T cell maturation stages, mainly CD4−CD8− and CD4+CD8+ thymocytes, as well as an increased presence of apoptotic cells, leading to significant thymic cortex reduction. Thymus atrophy showed no association with elevated serum cytokines levels, although increased glucocorticoid levels did. The severity of thymic damage in both models reached the same extend although it occurs at different stages of infection, showing that thymic atrophy does not depend on parasitemia level but on the specific host-parasite interaction.

Identification of a human blood biomarker of pharmacological 11β-hydroxysteroid dehydrogenase 1 inhibition.

11β-Hydroxysteroid dehydrogenase-1 (11β-HSD1) catalyses the oxoreduction of cortisone to cortisol, amplifying levels of active glucocorticoids. It is a pharmaceutical target in metabolic disease and cognitive impairments. 11β-HSD1 also converts some 7oxo-steroids to their 7β-hydroxy forms. A recent study in mice described the ratio of tauroursodeoxycholic acid (TUDCA)/tauro-7oxolithocholic acid (T7oxoLCA) as a biomarker for decreased 11β-HSD1 activity. The present study evaluates the equivalent bile acid ratio of glycoursodeoxycholic acid (GUDCA)/glyco-7oxolithocholic acid (G7oxoLCA) as a biomarker for pharmacological 11β-HSD1 inhibition in humans and compares it with the currently applied urinary (5α-tetrahydrocortisol + tetrahydrocortisol)/tetrahydrocortisone ((5αTHF + THF)/THE) ratio. Bile acid profiles were analysed by ultra-HPLC tandem-MS in blood samples from two independent, double-blind placebo-controlled clinical studies of the orally administered selective 11β-HSD1 inhibitor AZD4017. The blood GUDCA/G7oxoLCA ratio was compared with the urinary tetrahydro-glucocorticoid ratio for ability to detect 11β-HSD1 inhibition. No significant alterations were observed in bile acid profiles following 11β-HSD1 inhibition by AZD4017, except for an increase of the secondary bile acid G7oxoLCA. The enzyme product/substrate ratio GUDCA/G7oxoLCA was found to be more reliable to detect 11β-HSD1 inhibition than the absolute G7oxoLCA concentration in both cohorts. Comparison of the blood GUDCA/G7oxoLCA ratio with the urinary (5αTHF + THF)/THE ratio revealed that both successfully detect 11β-HSD1 inhibition. 11β-HSD1 inhibition does not cause major alterations in bile acid homeostasis. The GUDCA/G7oxoLCA ratio represents the first blood biomarker of pharmacological 11β-HSD1 inhibition and may replace or complement the urinary (5αTHF + THF)/THE ratio biomarker.

Acute or Chronic Exposure to Corticosterone Promotes Wakefulness in Mice.

Elevated glucocorticoid levels triggered by stress potentially contribute to sleep disturbances in stress-induced depression. However, sleep changes in response to elevated corticosterone (CORT), the major glucocorticoid in rodents, remain unclear. Here, we investigated the effects of acute or chronic CORT administration on sleep using electroencephalogram (EEG) and electromyography (EMG) recordings in freely moving mice. Acute CORT exposure rapidly promoted wakefulness, marked by increased episodes and enhanced EEG delta power, while simultaneously suppressing rapid eye movement (REM) and non-rapid eye movement (NREM) sleep, with the latter marked by decreased mean duration and reduced delta power. Prolonged 28-day CORT exposure led to excessive wakefulness and REM sleep, characterized by higher episodes, and decreased NREM sleep, characterized by higher episodes and reduced mean duration. EEG theta activity during REM sleep and delta activity during NREM sleep were attenuated following 28-day CORT exposure. These effects persisted, except for REM sleep amounts, even 7 days after the drug withdrawal. Elevated plasma CORT levels and depressive phenotypes were identified and correlated with observed sleep changes during and after administration. Fos expression significantly increased in the lateral habenula, lateral hypothalamus, and ventral tegmental area following acute or chronic CORT treatment. Our findings demonstrate that CORT exposure enhanced wakefulness, suppressed and fragmented NREM sleep, and altered EEG activity across all stages. This study illuminates sleep alterations during short or extended periods of heightened CORT levels in mice, providing a neural link connecting insomnia and depression.

Salivary corticosterone reflects plasmatic levels in a wild seabird

Wild animals have been increasingly exposed to a wide range of stressors, mainly due to the intensification of human activities and habitat modifications. Consequently, new tools in order to assess the physiological and health status of wild animals have been developed. In particular, glucocorticoids have received a special attention. Primarily metabolic hormones, they are also used to evaluate the stress level of organisms. While historically measured in blood samples, new less-invasive methods have been recently developed to measure glucocorticoids in matrices such as faeces, hairs/feathers, or saliva. To date, measurements in saliva are still in their infancy despite the numerous advantages of the matrix: non-invasive, reflects the biologically active portion of glucocorticoids, allows to measure both baseline and stress-induced levels. In addition, most studies using saliva have been performed on domestic and captive animals, and recent development in wild animals have focused on mammals. Here, we show, for the first time, that saliva could also be reliably used in free-ranging birds, as glucocorticoid levels in saliva strongly correlated with plasma levels. This promising result opens new avenues for a non-invasive sampling method to assess health status of wild birds in conservation biology and ecology.

Chemogenetic activation of CRF neurons as a model of chronic stress produces sex-specific physiological and behavioral effects

Trauma and chronic stress exposure are the strongest predictors of lifetime neuropsychiatric disease presentation. These disorders often have significant sex biases, with females having higher incidences of affective disorders such as major depression, anxiety, and PTSD. Understanding the mechanisms by which stress exposure heightens disease vulnerability is essential for developing novel interventions. Current rodent stress models consist of a battery of sensory, homeostatic, and psychological stressors that are ultimately integrated by corticotropin-releasing factor (CRF) neurons to trigger corticosteroid release. These stress paradigms, however, often differ between research groups in the type, timing, and duration of stressors utilized. These inconsistencies, along with the variability of individual animals’ perception and response to each stressor, present challenges for reproducibility and translational relevance. Here, we hypothesized that a more direct approach using chemogenetic activation of CRF neurons would recapitulate the effects of traditional stress paradigms and provide a high-throughput method for examining stress-relevant phenotypes. Using a transgenic approach to express the Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) receptor hM3Dq in CRF-neurons, we found that the DREADD ligand clozapine-N-oxide (CNO) produced an acute and robust activation of the hypothalamic-pituitary-adrenal (HPA) axis, as predicted. Interestingly, chronic treatment with this method of direct CRF activation uncovered a novel sex-specific dissociation of glucocorticoid levels with stress-related outcomes. Despite hM3Dq-expressing females producing greater corticosterone levels in response to CNO than males, hM3Dq-expressing males showed significant typical physiological stress sensitivity with reductions in body and thymus weights. hM3Dq-expressing females while resistant to the physiological effects of chronic CRF activation, showed significant increases in baseline and fear-conditioned freezing behaviors. These data establish a novel mouse model for interrogating stress-relevant phenotypes and highlight sex-specific stress circuitry distinct for physiological and limbic control that may underlie disease risk.

Exposure to ecologically relevant estrogen levels do not influence morphology or immune parameters in hatchling American alligators (Alligator mississippiensis)

Human activity has increased concentrations of endocrine-disrupting contaminants (EDCs) in many ecological systems. Many EDCs are xenoestrogens, which imitate naturally derived estrogen like estradiol 17-β (E2). These pollutants can critically affect a broad range of biological functions, particularly in organisms inhabiting aquatic environments. E2 and associated receptors are involved in regulating innate immune responses, where documentation of exogenous E2 on immune parameters is important for understanding health consequences. In this study, we explore the impact of environmentally relevant concentrations of E2 on circulating glucocorticoid levels and several innate immune parameters in hatchling American alligators (Alligator mississippiensis). Twenty-three hatchling alligators were randomly placed in one of three groups that differed in dietary E2 concentration: control (no E2 exposure), low E2 (0.5 μg/kg E2), or high E2 (1 μg/kg E2) for 10 weeks. Following this period, several biomarkers were quantified to monitor the impact of E2: growth, change in body condition, white blood cell (WBC) counts, glucocorticoid levels, and general antibody response. Blood E2 concentrations were greater in individuals exposed to E2, but plasma corticosterone levels were reduced among the experimental groups. Morphology, growth, and immune parameters of E2 exposed animals did not differ from controls. These results suggest that acute exposure to increased environmental estrogen concentrations may alter plasma hormone concentrations but have little to no impact on immediate morphology or immune responses. Future studies may expand on this by monitoring biomarkers in wild populations across time, which will provide insight into how different ontogenetic stages are impacted by environmental contaminants.

SAT011 Neonatal Endotoxin Exposure Has Long-term Effects On Glucocorticoid Regulation In Blood And Lymphoid Organs Of Mice

Abstract Disclosure: A. Mazurenko: None. M. Salehzadeh: None. S.R. Lim: None. K.K. Soma: None. Glucocorticoids (GCs) are steroid hormones that play vital roles in immunity. GCs are produced by the adrenal glands and also by lymphoid organs, such as bone marrow, thymus, and spleen. There is a period of decreased adrenocortical activity in neonatal humans and mice termed the stress hyporesponsive period (SHRP), which protects developing tissues from high circulating GCs and allows for fine-tuning of GC levels in lymphoid organs. In neonatal mice, GC levels increase acutely in response to the bacterial endotoxin, lipopolysaccharide (LPS). Yet, it is not known how early-life LPS exposure affects systemic and local GC levels in adulthood. We assessed whether LPS administration during the SHRP (‘first hit’) affects GC regulation in blood and lymphoid organs in adulthood when challenged again with LPS (‘second hit’). We administered LPS (50 μg/kg i.p.) or saline (vehicle control) to male and female C57BL/6J neonatal mice (PND 4 and 6, within the SHRP). We then split the mice into two groups and administered LPS (50 μg/kg i.p.) or saline to adults (PND90) (2x2 design). We collected whole blood, bone marrow, thymus, and spleen 4 hr after LPS administration at PND90. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure 7 steroids, including 11-deoxycorticosterone (DOC; corticosterone precursor), corticosterone (main active GC in mice), and 11-dehydrocorticosterone (DHC; corticosterone metabolite). Preliminary results show that neonatal LPS treatment altered LPS-stimulated corticosterone levels (but not baseline corticosterone levels) in adult mice. Specifically, neonatal LPS treatment increased blood corticosterone levels in LPS-treated adult males, but not females. In contrast, neonatal LPS treatment increased thymus and spleen corticosterone levels in LPS-treated adult females. There were no effects of neonatal LPS treatment on DOC and DHC levels in adult males. In contrast, neonatal LPS treatment increased thymus DOC and spleen DHC levels in LPS-treated adult females. Altogether, our results suggest that LPS exposure during the SHRP has long-term effects on GC regulation in the blood and lymphoid organs when challenged with a ‘second hit’ in adulthood. This research offers vital insights into understanding the development and regulation of the immune and endocrine systems. Critically, this study demonstrates that lymphoid organs modulate local GC levels independently of circulating GC levels. More generally, these data elucidate the long-lasting programming effects of an immunological stressor, such as neonatal sepsis, early in life. Presentation: Saturday, June 17, 2023

OR19-01 Srd5a1 Undergoes Distinct Changes in Methylation and Is Differentially Expressed During Pre-Pubertal Development in the Ovary and Hypothalamus

Abstract Disclosure: B. Bar-Sadeh: None. P. Melamed: None. The steroidogenic enzyme, 5α-reductase-1, is responsible for conversion of testosterone to dihydrotestosterone, and production of other 5α-reduced steroids and their derivatives, including in the brain. We reported previously that Srd5a1 levels are reduced in mice hypothalamic preoptic area (POA) and ovaries following early-life immune stress, which appeared to underlie delayed puberty onset and faster depletion of the ovarian reserve. This drop in ovarian Srd5a1 expression was concomitant with hypermethylation at two CpGs in its first intron, and also at the orthologous site in buccal DNA of women who experienced childhood immune challenge1. We thus hypothesized that this region of Srd5a1 acts as a transcriptional enhancer and is regulated by DNA methylation. Here we examined Srd5a1 expression and methylation at these CpGs across normal mouse development and in cultured cells. Ovarian Srd5a1 increased 8-fold and CpG methylation decreased by as much as 75% between PND10-30. During this time estradiol (E2) levels rise, and E2 exposure increased Srd5a1 mRNA levels in primary ovarian cells and KK-1 cell line. Chromatin immunoprecipitation (ChIP) confirmed binding of estrogen receptor to the locus of the CpGs, and identified an adjacent region strongly enriched with H3K4me1, characteristic of transcriptional enhancers. In the POA, in sharp contrast, Srd5a1 mRNA levels decreased by 70% between PND7-10 and then stayed quite constant, with little correlation to CpG methylation levels. The neonatal drop in POA Srd5a1 coincides with an increase in glucocorticoid (GC) levels, and we had seen lower Srd5a1 expression in mice following early-life stress, we thus treated GT1-7 hypothalamic neuronal cells with dexamethasone. This treatment reduced Srd5a1 expression, and ChIP confirmed that the GC receptor binds the enhancer locus. Srd5a1 expression was 12-fold higher in the ovarian KK1 cells compared to the neuronal GT1-7 cells, in accordance with much higher methylation levels in the latter. We thus used these cells to investigate the role of this CpG methylation, using gRNAs targeting dCas9-TET1 for demethylation in GT1-7 cells, or dCas9-DNMT3 for inducing methylation in KK1 cells. In GT1-7 cells, methylation at the first CpG dropped >50%, though was unaltered at the second CpG, and no effect was seen on Srd5a1 expression or its response to E2. In contrast, methylation in KK1 cells increased at the second CpG only, by more than 2.5 fold, and this abolished the response to E2. These distinct changes in Srd5a1 expression during development, together with our findings in cultured cells suggest a cell-specific GC-activated repression of Srd5a1 in the POA, and its E2-activated stimulation in the ovaries regulated by methylation at one of these CpGs. Reference: 1. Bar-Sadeh, B., Amichai, O.E., Pnueli, L., Begum, K., Leeman, G., Emes, R.D., Stöger, R., Bentley, G.R., and Melamed, P. (2022). BMC Biol. 20, 11. Presentation Date: 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.

Technical validation and a comparison of two methods to quantify individual levels of glucocorticoids in Alpine marmot hair

Quantification of cortisol concentration in hair has become a promising conservation tool for non-invasive monitoring of “stress” in wild populations, yet this method needs to be carefully validated for each species. The goals of the study were:•Immunologically validate two methods (study 1 and 2 respectively) to extract and quantify cortisol in the hair of wild Alpine marmots.•Compare the amount of cortisol extracted from hair samples using two methods i.e. cut into fine pieces (study 1) and hair samples pulverized using a ball mill (study 2).•Determine the extent to which methods in study 2 could provide individual specific hair cortisol (HC) measures when samples were taken from the same body location. Within and between individual variations in HC levels were examined from multiple hair samples from 14 subjects in study 2. We evaluated if inter-individual variations in HC levels could be explained by sex and age.At least twice the amount of cortisol was obtained per g/hair when samples were pulverized in a ball mill prior to extraction compared to when cut into pieces. Our methods demonstrated intra-individual consistency in HC at a given time point: inter-individual variation in HC was three times larger than within individual variance. Sex and age did not impact HC levels.

FRI176 Integrated Profiling Analysis Of Serum Steroids And Catecholamines In A Single Run With LC-MS

Abstract Disclosure: J. Noh: None. C. Lee: None. S. Park: None. C. Ahn: None. S. Myung: None. J. Kim: None. M. Choi: None. Adrenal steroids and catecholamines are associated with the response against biochemical stress and adrenal disorders. Metabolic alteration of these hormones could be quantitatively evaluated in diagnostic process, but both adrenal cortex and medullary hormones have been measured in different analytical platforms. To increase the bio-analytical validity, liquid chromatography-mass spectrometry (LC-MS) based simultaneous profiling of 29 steroids and 6 catecholamines was developed. After alkyloxycarbonylation with isobutyl chloroformate, catecholamines and steroids were extracted simultaneously in liquid-liquid extraction. The limit of quantification ranged from 0.1 to 25 ng/mL with extraction recoveries of 78.5-97.0%, while the precision and accuracy were 4.8-7.9% and 98.0-110.9%, respectively. The validated LC-MS assay was then applied to serum samples obtained from 20 individual patients with primary aldosteronism (PA), Cushing’s syndrome (CS) and pheochromocytoma and paraganglioma (PPGL). The significantly increased serum levels of 18-hydroxycorticosterone (p < 0.005), corticosterone (p < 0.05), and 18-hydroxycortisol (p < 0.002) were observed in PA group, while PPGL group showed decreased level of 11-deoxycortisol (p < 0.007). Higher levels of glucocorticoids including 21-deoxycortisol (p < 0.002), 6β-hydroxycortisol (p < 0.001), and tetrahydrocortisone (p < 0.001) and lower levels of androgens including DHEA (p < 0.00001) and DHEA sulfate (p < 0.03) were detected in CS group. Catecholamines, such as norepinephrine (p < 0.0001) and normetanephrine (p < 0.0001), were significantly increased in PPGL patients. The present novel assay was successfully applied to discriminate three different adrenal diseases in a single analytical run. Presentation: Friday, June 16, 2023

Blunted Glucocorticoid Responsiveness to Stress Causes Behavioral and Biological Alterations That Lead to Posttraumatic Stress Disorder Vulnerability

BackgroundUnderstanding why only a subset of trauma-exposed individuals develop posttraumatic stress disorder is critical for advancing clinical strategies. A few behavioral (deficits in fear extinction) and biological (blunted glucocorticoid levels, small hippocampal size, and rapid-eye-movement sleep [REMS] disturbances) traits have been identified as potential vulnerability factors. However, whether and to what extent these traits are interrelated and whether one of them could causally engender the others are not known. MethodsIn a genetically selected rat model of reduced corticosterone responsiveness to stress, we explored posttraumatic stress disorder–related biobehavioral traits using ex vivo magnetic resonance imaging, cued fear conditioning, and polysomnographic recordings combined with in vivo photometric measurements. ResultsWe showed that genetic selection for blunted glucocorticoid responsiveness led to a correlated multitrait response, including impaired fear extinction (observed in males but not in females), small hippocampal volume, and REMS disturbances, supporting their interrelatedness. Fear extinction deficits and concomitant disruptions in REMS could be normalized through postextinction corticosterone administration, causally implicating glucocorticoid deficiency in two core posttraumatic stress disorder–related risk factors and manifestations. Furthermore, reduced REMS was accompanied by higher norepinephrine levels in the hippocampal dentate gyrus that were also reversed by postextinction corticosterone treatment. ConclusionsOur results indicate a predominant role for glucocorticoid deficiency over the contribution of reduced hippocampal volume in engendering both REMS alterations and associated deficits in fear extinction consolidation, and they causally implicate blunted glucocorticoids in sustaining neurophysiological disturbances that lead to fear extinction deficits.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Minireview: Glucocorticoid-Leptin Crosstalk: Role of Glucocorticoid-Leptin Counterregulation in Metabolic Homeostasis and Normal Development.

Glucocorticoids and leptin are two important hormones that regulate metabolic homeostasis by controlling appetite and energy expenditure in adult mammals. Also, glucocorticoids and leptin strongly counterregulate each other, such that chronic stress-induced glucocorticoids upregulate the production of leptin and leptin suppresses glucocorticoid production directly via action on endocrine organs and indirectly via action on food intake. Altered glucocorticoid or leptin levels during development can impair organ development and increase the risk of chronic diseases in adults, but there are limited studies depicting the significance of glucocorticoid-leptin interaction during development and its impact on developmental programming. In mammals, leptin-induced suppression of glucocorticoid production is critical during development, where leptin prevents stress-induced glucocorticoid production by inducing a period of short-hyporesponsiveness when the adrenal glands fail to respond to certain mild to moderate stressors. Conversely, reduced or absent leptin signaling increases glucocorticoid levels beyond what is appropriate for normal organogenesis. The counterregulatory interactions between leptin and glucocorticoids suggest the potential significant involvement of leptin in disorders that occur from stress during development.

Development programming: Stress during gestation alters offspring development in sheep.

Inappropriate management practices of domestic animals during pregnancy can be potential stressors, resulting in complex behavioural, physiological and neurological consequences in the developing offspring. Some of these consequences can last into adulthood or propagate to subsequent generations. We systematically summarized the results of different experimental patterns using artificially increased maternal glucocorticoid levels or prenatal maternal physiological stress paradigms, mediators between prenatal maternal stress (PMS)and programming effects in the offspring and the effects of PMS on offspring phenotypes in sheep. PMS can impair birthweight, regulate the development of the hypothalamic-pituitary-adrenal axis, modify behavioural patterns and cognitive abilities and alter gene expression and brain morphology in offspring. Further research should focus on the effects of programming on gene expression, immune function, gut microbiome, sex-specific effects and maternal behaviour of offspring, especially comparative studies of gestational periods when PMS is applied, continual studies of programming effects on offspring and treatment strategies that effectively reverse the detrimental programming effects of prenatal stress.