Determination of Fecal Glucocorticoid Metabolites to Evaluate Stress Response in <Emphasis Type="Italic">Alouatta pigra</Emphasis>

Measuring fecal glucocorticoid metabolites is now a common practice to assess the stress response in primates. Nevertheless, it is important to validate the utilized immunoassay for each primate species before the technique is applied to populations in the wild. We determined the stress response of black howlers (Alouatta pigra) via 2 different group-specific enzyme immunoassays (EIAs). 11-oxoetiocholanolone EIAs are suited to assess the stress response of black howlers via fecal glucocorticoid metabolites. Levels of fecal glucocorticoid metabolites increased after we applied a stressor, i.e. anesthesia, reaching peak concentrations 24–96 h poststressor. Both basal and stress-induced fecal glucocorticoid metabolite levels showed individual variations. The increase of fecal glucocorticoid metabolites after the stressor (paralleling increases in serum) indicates that one can effectively measure adrenocortical activity in Alouatta pigra via these 2 enzyme immunoassays. However, it is important to consider individual variations in the excretion of fecal glucocorticoid metabolites when planning field endocrinological research on Alouatta pigra. Fecal glucocorticoid metabolite excretion takes 1–3 d poststressor depending on the individual. Further, there is an important individual variability in the concentrations of glucocorticoid metabolites, which might reflect differences in stress reactivity or fecal glucocorticoid metabolite metabolism and excretion.     

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.     

Heritable variation in circulating glucocorticoids and endocrine flexibility in a free‐living songbird

Phenotypic flexibility is a central way that organisms cope with challenging and changing environments. As endocrine signals mediate many phenotypic traits, heritable variation in hormone levels, or their context‐dependent flexibility, could present an important target for selection. Several studies have estimated the heritability of circulating glucocorticoid levels under acute stress conditions, but little is known about the potential for either baseline hormone levels or rapid endocrine flexibility to evolve. Here, we assessed the potential for selection to operate on the elevation (circulating hormone levels) and flexibility of glucocorticoid reaction norms to acute restraint stress. Multivariate animal models revealed low but significant heritability in baseline (h² = 0.13–0.14) and stress‐induced glucocorticoids (h² = 0.18), and moderate heritability in glucocorticoid flexibility in response to acute stress (h² = 0.38) in free‐living juvenile tree swallows (Tachycineta bicolor; n = 408). Baseline glucocorticoids were not genetically correlated with either stress‐induced glucocorticoids or glucocorticoid flexibility. These findings indicate that baseline glucocorticoids and the acute stress response are distinct traits that can be independently shaped by selection. Microevolutionary changes that influence the expression or flexibility of these endocrine mediators of phenotype may be an important way that populations adapt to changing environments and novel threats.     

Dosing schedule-dependent attenuation of dexamethasone-induced muscle atrophy in mice

Many inflammatory and autoimmune diseases are treated using synthetic glucocorticoids. However, excessive glucocorticoid can often cause unpredictable effects including muscle atrophy. Endogenous glucocorticoid levels robustly fluctuate in a circadian manner and peak just before the onset of the active phase in both humans and nocturnal rodents. The present study determines whether muscle atrophy induced by exogenous glucocorticoid can be avoided by optimizing dosing times. We administered single daily doses of the glucocorticoid analog dexamethasone (Dex) to mice for 10 days at the times of day corresponding to peak (early night) or trough (early morning) endogenous glucocorticoid levels. Administration at the acrophase of endogenous glucocorticoids significantly attenuated Dex-induced wasting of the gastrocnemius (Ga) and tibialis anterior (TA) muscles that comprise mostly fast-twitch muscle fibers. Real-time RT-PCR revealed that the Dex-induced mRNA expression of genes encoding the atrophy-related ubiquitin ligases Muscle Atrophy F-box (Fbxo32, also known as MAFbx/Atrogin-1) and Muscle RING finger 1 (Trim63, also known as MuRF1) in the Ga and TA muscles was significantly attenuated by Dex when administered during the early night. Dex negligibly affected the weight of the soleus (So) muscle that mostly comprises slow-twitch muscle fibers, but significantly and similarly decreased the weight of the spleen at both dosing times. These results suggest that glucocorticoid-induced muscle atrophy can be attenuated by optimizing the dosing schedule.     

Intracerebroventricular Glucocorticoid Infusion in Normal Rats: Induction of Parasympathetic‐Mediated Obesity and Insulin Resistance

Objective: The aims of the present study were to determine whether increased body weight resulting from intracerebroventricular (ICV) glucocorticoid (dexamethasone) infusion in normal rats is associated, as in obesity, with changes in glucose metabolism and to investigate whether the parasympathetic nervous system is involved in the glucocorticoid‐induced effects. Research Methods and Procedures: Male Sprague–Dawley rats were infused with ICV dexamethasone (2.5 μg/d) or its vehicle for 2 days during which food intake, body weight, and basal insulinemia were measured. Euglycemic–hyperinsulinemic clamps associated with the labeled 2‐deoxyglucose technique were then performed to determine the total rate of glucose disappearance and the tissue glucose use indices. Similar experiments were carried out in vagotomized rats. Results: Two days of ICV glucocorticoid infusion in normal rats resulted in increases in food intake, body weight, basal insulinemia, and produced decreases in the insulin‐stimulated total rate of glucose disappearance, as well as in glucose use indices of all muscle types studied. None of these alterations was observed when glucocorticoid infusion was carried out in vagotomized rats. Discussion: These data show that central glucocorticoid infusion favors anabolic processes, such as feeding behavior, body weight gain, and insulin output, while promoting muscle insulin resistance. These effects seem to be mediated by an activation of the parasympathetic nervous system, because they all disappear when tested in vagotomized rats.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone‐Induced Muscle Atrophy In Vitro

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid‐induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid‐induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel‐like factor 15 (KLF15). Heat stress recovered the dexamethasone‐induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress‐induced protection against glucocorticoid‐induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650–664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.     

Shyness–Boldness,but not Exploration,Predicts Glucocorticoid Stress Response in Richardson's Ground Squirrels (Urocitellus richardsonii)

The relationship between stress and personality has often been studied using captive animals in a laboratory context, yet less often in wild populations. Wild populations, however, may reveal aspects of the personality–stress relationship that laboratory‐based studies cannot. Here, we assessed the personality and stress hormone response of adult females within a free‐living population of Richardson's ground squirrels (Urocitellus richardsonii). Personality was assessed by quantifying individual responses to a novel object, and physiological stress was measured from faecal glucocorticoid metabolites. Principal component and principal component regression analyses were performed to determine whether the behavioural and endocrine measures were related. Based on these analyses, shyness–boldness was found to best predict glucocorticoid levels, in that individuals expressing the greatest vigilance in response to the novel object also had the highest measured concentrations of faecal glucocorticoids. Exploration, however, was independent of measured glucocorticoid levels, consistent with a multidimensional interpretation of non‐human animal personality.     

Evolutionary conservation of an atypical glucocorticoid‐responsive element in the human tyrosine hydroxylase gene

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at ?7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein‐1 (AP‐1)‐like motif in the rat TH gene. We cloned this hTH‐CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH‐CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5′‐TGACTAA at ?7243 bp completely abolished the Dex‐stimulated Luc activity of hTH‐CRII construct. The AP‐1 agonist, tetradeconoyl‐12,13‐phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP‐1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid‐responsive element in a 7 bp AP‐1‐like motif in the promoter region at ?7.24 kb of the human TH gene.     

Resource allocation in Wilson's storm-petrels<Emphasis Type="Italic"> Oceanites oceanicus</Emphasis> determined by measurement of glucocorticoid excretion

When resources are limited, life-history theory predicts that long-lived animals should allocate available resources to body maintenance rather than to reproduction in order to maximise their lifetime reproductive success. In the present study, we estimated physiological stress in a small procellariiform seabird, the Wilson's storm-petrel Oceanites oceanicus, as a means of understanding how limited resources are partitioned between provisioning parents and their chicks. We analysed adrenocortical activity of Wilson's storm-petrels during the breeding season by measuring glucocorticoid (GC) excretion, using an enzyme immunoassay measuring tetrahydrocorticosterone concentrations in extracts of faeces and urine of chicks and adults. Faecal GC measures were negatively correlated with chick body condition, suggesting that measures of tetrahydrocorticosterone in faeces and urine can be used to assess adrenal activity characteristic for physiological stress in Wilson's storm-petrels. In the breeding season of 1999, the colony was subject to low food availability, and the faecal and urine GC levels of chicks were elevated during these months of chronic starvation. In contrast, adults did not show elevated GC levels. The data thus suggest that Wilson's storm-petrels respond to unfavourable conditions by maintaining their own body condition and reducing provisioning of food to their chicks. Communicated by R.F. Oliviera     

Seasonal dynamics of agonistic behavior and hormones in an ex situ all‐male colony of large flying foxes

Large flying foxes (Pteropus vampyrus) are a socially complex species. In situ colonies typically comprise thousands of individuals in small harems of one male to many females. In ex situ environments, all‐male colonies are becoming more common due to a surplus of males in the population. There is limited information describing the hormonal and behavioral patterns of all‐male colonies during the breeding season. We assessed seasonal changes in hormones and behavior in an all‐male colony of 12 large flying foxes at Disney's Animal Kingdom^®. We validated hormone assays using morning urine and fecal samples to assess seasonal changes in excreted immunoreactive testosterone and glucocorticoid metabolites. We collected behavior data using an all‐occurrence method, recording agonistic behaviors related to territorial defense (hooking, biting, wing flexing, vocalizing, and wrestling), and sexual behavior (mounting and frontal grabbing). Results indicated that (i) we could reliably measure testosterone and glucocorticoid metabolites concentrations from fecal and urine samples collected from individual bats; (ii) there were distinct relationships between changes in levels of agonism and hormone concentrations throughout the year; and (iii) three agonistic behaviors (chasing, wrestling, and open‐mouth threat) peaked prior to the increase in testosterone and glucocorticoid hormones measured during the breeding season. These three behaviors could potentially be used as early indicators to signal the onset of the breeding season and allow time to implement ex situ management changes to reduce the incidence of agonism between individuals.     

Stress Response of Working African Elephants to Transportation and Safari Adventures

Abstract: African elephants (Loxodonta africana) are intensively managed in southern Africa and are routinely translocated between reserves. Domesticated elephants are used for elephant-back safaris and interactions with guests. Understanding how elephants respond to such activities is critical because of welfare issues associated with both humans and elephants. We investigated the stress response (i.e., fecal glucocorticoid metabolite secretion [FGM]) of working elephants in Letsatsing Game Reserve, South Africa, over 1 year to evaluate their response to transportation and ecotourism activities. We used free-ranging elephants in adjacent Pilanesburg National Park as controls. Fecal glucocorticoid metabolites were greatest prior to and during translocation and declined over the year. Within 1–2 months of transportation, FGM levels in working elephants became indistinguishable from those in wild elephants. Fecal glucocorticoid metabolite levels were higher during human interaction days than days without interaction. The highest observed FGM levels were associated with transportation and episodic loud noises. Transportation is a stressful activity for elephants, and ≥3 months should be provided to translocated elephants to acclimate to their new surroundings. Although stress levels of elephants increased slightly when interacting with humans in the contexts we studied, evaluating interactions under a wider range of contexts is necessary to minimize danger to elephants and humans.     

Light interference and melatonin affects digestion and glucocorticoid metabolites in striped mouse

Animals secrete glucocorticoids to deal with daily stressors. Studies have found that supplemental melatonin decreases glucocorticoid metabolite levels in stressed animals. We determined the effect of light interference (LI) and supplemental melatonin on (1) body mass, (2) food intake and (3) glucocorticoid metabolite levels of the striped mouse (Rhabdomys pumilio). Experiment was split into three phases: 8 L: 16 D; 8 L: 16 D with a 15 min light interruption every 4 h; and 8 L: 16 D with a 15 min light interruption every 4 h and melatonin (0.2 μg/ml) added to the water. Body mass was significantly different between phases with lowest body mass (89.17 ± 6.56 g) occurring during standard 8 L: 16 D. LI and melatonin significantly increased body mass. LI increased and melatonin decreased glucocorticoid metabolite levels. LI significantly increased and melatonin significantly decreased assimilation efficiencies possibly due to changes in energetic demands.     

Absence of Hypersensitivity to Glucocorticoids in Antiretroviral‐associated Lipodystrophy

Objective: The lipodystrophy syndrome, which is associated with the use of antiretroviral drugs in some human immunodeficiency virus (HIV)‐infected individuals, bears a striking similarity to the fat redistribution observed in Cushing's disease. Although urinary free cortisol excretion and glucocorticoid receptor binding affinity are not elevated in subjects with lipodystrophy, glucocorticoid action at the cellular level has not been examined in affected individuals. The objective of this study was to determine whether tissue sensitivity to glucocorticoids is increased in subjects with lipodystrophy taking protease inhibitors. Research Methods and Procedures: Subjects included 11 HIV‐infected men on protease inhibitor therapy with lipodystrophy and 10 control HIV‐infected men not on protease inhibitor therapy and without lipodystrophy. Trunk to extremity fat ratio was measured by DXA. Dexamethasone suppression of peripheral blood mononuclear cell proliferation was measured as an index of tissue sensitivity to glucocorticoid action. Results: Compared with the control group, subjects with lipodystrophy had a significant elevation of the trunk to extremity fat ratio [median (interquartile range): 2.9 (1.3) vs. 1.6 (1.2); p < 0.05]. The concentration of dexamethasone resulting in 50% maximal suppression of proliferation was 11.7 nM (9.3 nM) in subjects with lipodystrophy and 19.6 nM (9.7 nM) in control subjects (p = not significant), and the percentage minimal proliferation was 4% (12%) and 17% (18%) in the two groups, respectively (p = not significant). Discussion: Despite the Cushingoid appearance of affected individuals, these data suggest that body fat redistribution in antiretroviral‐associated lipodystrophy does not arise through an increase in postreceptor glucocorticoid signaling.     

A non-invasive method for measuring glucocorticoid metabolites (GCM) in Mountain hares (<Emphasis Type="Italic">Lepus timidus</Emphasis>)

In this study, we evaluated a non-invasive method for measuring glucocorticoid metabolites (GCM) in the Mountain hare (Lepus timidus). An adrenocorticotrophic hormone challenge test was performed in order to select an appropriate enzyme immunoassay (EIA) to measure faecal GCM. Finally, an 11-oxoaetiocholanolone EIA and a 5α-pregnane-3β,11β,21-triol-20-one EIA were chosen. Both assays showed small fluctuations in baseline values and a clear response after stimulation of the adrenocortical activity. To test the stability of faecal metabolites under field conditions, the effects of different storage conditions and periods on GCM concentrations were examined. The assays revealed low fluctuations in metabolite concentrations within the storage period of 12, 24, 48 and 72 h, both at ambient temperatures of 10°C and 25°C, respectively. A washing-out effect of water was found for both assays, which must be taken into account in field studies. The results indicate that this non-invasive method can be used to evaluate glucocorticoid levels of free-ranging Mountain hares.     

Dynamics of glucocorticoids in the ontogenesis of freshwater crawfish Astacus leptodactylus Esch

Dynamics of changes in glucocorticoid (hydrocortisone and corticosterone) levels was studied in the ontogenesis of freshwater crawfish Astacus Leptodactylus Esch. It was shown that steroid concentrations increase during the embryogenesis period. Decrease in the glucocorticoid levels during postembryonic development is most probably related to the stabilization of young fish growth and the attainment of hormone levels typical for adult individuals. The correlation ofglucocorticoid levels with physiological-biochemical changes at different stages of ontogenesis indicates that these steroids play an essential role in the regulation of freshwater crawfish vital functions.     

Environmental Enrichment Effect on Fecal Glucocorticoid Metabolites and Captive Maned Wolf (Chrysocyon brachyurus) Behavior

Environmental enrichment is a technique that may reduce the stress of nonhuman animals in captivity. Stress may interfere with normal behavioral expression and affect cognitive decision making. Noninvasive hormonal studies can provide important information about the stress statuses of animals. This study evaluated the effectiveness of different environmental enrichment treatments in the diminution of fecal glucocorticoid metabolites (stress indicators) of three captive maned wolves (Chrysocyon brachyurus). Correlations of the fecal glucocorticoid metabolite levels with expressed behaviors were also determined. Results showed that environmental enrichment reduced fecal glucocorticoid metabolite levels. Furthermore, interspecific and foraging enrichment items were most effective in reducing stress in two of the three wolves. No definite pattern was found between behavioral and physiological responses to stress. In conclusion, these behavioral and physiological data showed that maned wolves responded positively from an animal well being perspective to the enrichment items presented.