Erin Faught

Rapid cortisol signaling in response to acute stress involves changes in plasma membrane order in rainbow trout liver

The activation of genomic signaling in response to stressor-mediated cortisol elevation has been studied extensively in teleosts. However, very little is known about the rapid signaling events elicited by this steroid. We tested the hypothesis that cortisol modulates key stress-related signaling pathways in response to an acute stressor in fish liver. To this end, we investigated the effect of an acute stressor on biophysical properties of plasma membrane and on stressor-related protein phosphorylation in rainbow trout (Oncorhynchus mykiss) liver. A role for cortisol in modulating the acute cellular stress response was ascertained by blocking the stressor-induced elevation of this steroid by metyrapone. The acute stressor exposure increased plasma cortisol levels and liver membrane fluidity (measured by anisotropy of 1,6-diphenyl-1,3,5-hexatriene), but these responses were abolished by metyrapone. Atomic force microscopy further confirmed biophysical alterations in liver plasma membrane in response to stress, including changes in membrane domain topography. The changes in membrane order did not correspond to any changes in membrane fatty acid components after stress, suggesting that changes in membrane structure may be associated with cortisol incorporation into the lipid bilayer. Plasma cortisol elevation poststress correlated positively with activation of intracellular stress signaling pathways, including increased phosphorylation of extracellular signal-related kinases as well as several putative PKA and PKC but not Akt substrate proteins. Together, our results indicate that stressor-induced elevation of plasma cortisol level is associated with alterations in plasma membrane fluidity and rapid activation of stress-related signaling pathways in trout liver.

Maternal stress-associated cortisol stimulation may protect embryos from cortisol excess in zebrafish

Abnormal embryo cortisol level causes developmental defects and poor survival in zebrafish (Danio rerio). However, no study has demonstrated that maternal stress leads to higher embryo cortisol content in zebrafish. We tested the hypothesis that maternal stress-associated elevation in cortisol levels increases embryo cortisol content in this asynchronous breeder. Zebrafish mothers were fed cortisol-spiked food for 5 days, to mimic maternal stress, followed by daily breeding for 10 days to monitor temporal embryo cortisol content. Cortisol treatment increased mean embryo yield, but the daily fecundity was variable among the groups. Embryo cortisol content was variable in both groups over a 10-day period. A transient elevation in cortisol levels was observed in the embryos from cortisol-fed mothers only on day 3, but not on subsequent days. We tested whether excess cortisol stimulates 11βHSD2 expression in ovarian follicles as a means to regulate embryo cortisol deposition. Cortisol treatment in vitro increased 11β HSD2 levels sevenfold, and this expression was regulated by actinomycin D and cycloheximide suggesting tight regulation of cortisol levels in the ovarian follicles. We hypothesize that cortisol-induced upregulation of 11βHSD2 activity in the ovarian follicles is a mechanism restricting excess cortisol incorporation into the eggs during maternal stress.

Novel Nongenomic Signaling by Glucocorticoid May Involve Changes to Liver Membrane Order in Rainbow Trout

Stress-induced glucocorticoid elevation is a highly conserved response among vertebrates. This facilitates stress adaptation and the mode of action involves activation of the intracellular glucocorticoid receptor leading to the modulation of target gene expression. However, this genomic effect is slow acting and, therefore, a role for glucocorticoid in the rapid response to stress is unclear. Here we show that stress levels of cortisol, the primary glucocorticoid in teleosts, rapidly fluidizes rainbow trout (Oncorhynchus mykiss) liver plasma membranes in vitro. This involved incorporation of the steroid into the lipid domains, as cortisol coupled to a membrane impermeable peptide moiety, did not affect membrane order. Studies confirmed that cortisol, but not sex steroids, increases liver plasma membrane fluidity. Atomic force microscopy revealed cortisol-mediated changes to membrane surface topography and viscoelasticity confirming changes to membrane order. Treating trout hepatocytes with stress levels of cortisol led to the modulation of cell signaling pathways, including the phosphorylation status of putative PKA, PKC and AKT substrate proteins within 10 minutes. The phosphorylation by protein kinases in the presence of cortisol was consistent with that seen with benzyl alcohol, a known membrane fluidizer. Our results suggest that biophysical changes to plasma membrane properties, triggered by stressor-induced glucocorticoid elevation, act as a nonspecific stress response and may rapidly modulate acute stress-signaling pathways.

Mechanisms of cortisol action in fish hepatocytes

Here we provide an overview of the mechanistic characterization of the hepatic action of cortisol during stress in fish. Cortisol is the main circulating glucocorticoid in fish and its action is mediated through its cytosolic receptor, the glucocorticoid receptor (GR), and regulates the expression of genes involved in growth, metabolism and immune function. When taken together, the data suggests that cortisol may be playing a key role in the energy substrate re-partitioning in hepatocytes to cope with stress. The proposed model is that cortisol upregulates pathways involved in energy substrate mobilization, including gluconeogenesis, while downregulating energy demanding pathways, including growth and immune function. Recent work also points to a role for cortisol in mediating rapid action that is non-genomic and includes modulation of secondary signalling cascades; however, the physiological relevance of these studies remains to be determined. Altogether, studies carried out in hepatocytes are bringing to fore the complex nature of the cortisol signalling pathways in the organismal stress response. The mode of actions and their physiological implications for stress coping awaits further study.

The Molecular Stress Response

1. Introduction 2. Molecular Regulation of the Hypothalamic–Pituitary–Interrenal (HPI) Axis 2.1. Hypothalamus 2.2. Pituitary 2.3. Head Kidney (Interrenal Tissue) 3. Genomic Cortisol Signaling 3.1. Glucocorticoid Receptor 3.2. Mineralocorticoid Receptor 4. Genomic Effects of Cortisol 4.1. Development of the Stress Axis 4.2. Molecular Adjustments During Stress 4.3. Cellular Adjustments 5. Significance of Molecular Responses 6. Approaches to Study Molecular Responses to Stress 6.1. Mechanistic Studies Using Targeted Mutagenesis 6.2. Epigenetic Regulation of Stress Response 7. Concluding Remarks and the Unknowns In this chapter we summarize the key patterns observed in the transcript abundances of genes involved in the hypothalamic–pituitary–interrenal (HPI) axis regulation, as well as regulation of stress-responsive genes that are modulated by corticosteroid signaling in fish. Plasma levels of cortisol, the primary corticosteroid in teleosts, rise within minutes after a stressor encounter, and this steroid action is mediated primarily by genomic signaling involving the family of nuclear receptors including the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Specific molecular responses in target tissues associated with activation of these corticosteroid receptors have become apparent by increased use of receptor antagonists and gene knockdown tools. Although molecular adjustments to stress are dependent on species, developmental stage, type, and duration of stressor, overall cortisol action limits energy-demanding processes and enhances energy mobilization and reallocation. Recent work has also underscored the necessity of maternal cortisol and GR signaling as essential for fish development, but the underlying mechanisms are far from clear. While transcript abundance is an excellent indicator of pathway modulation by cortisol, this data by itself lacks physiological relevance unless accompanied by downstream protein and/or metabolite changes. As the field of molecular biology continues to advance, approaches including next-generation sequencing and gene editing tools will allow production of transgenic animals, even in nonmodel fish species, to reveal molecular mechanisms essential for stress adaptation. This fundamental knowledge may have relevance to improving the welfare of the organism in aquaculture and for protecting ecosystem health and biodiversity.

Plasma exosomes are enriched in Hsp70 and modulated by stress and cortisol in rainbow trout

Exosomes are endosomally derived vesicles that are secreted from cells and contain a suite of molecules, including proteins and nucleic acids. Recent studies suggest the possibility that exosomes in circulation may be affecting recipient target cell function, but the modes of action are unclear. Here, we tested the hypothesis that exosomes are in circulation in fish plasma and that these vesicles are enriched with heat shock protein 70 (Hsp70). Exosomes were isolated from rainbow trout (Oncorhynchus mykiss) plasma using differential centrifugation, and their presence was confirmed by transmission electron microscopy and the exosomal marker acetylcholinesterase. Plasma exosomes were enriched with Hsp70, and this stress protein was transiently elevated in trout plasma in response to a heat shock in vivo Using trout hepatocytes in primary culture, we tested whether stress levels of cortisol, the principle corticosteroid in teleosts, regulates exosomal Hsp70 content. As expected, a 1-h heat shock (+15°C above ambient) increased Hsp70 expression in hepatocytes, and this led to higher Hsp70 enrichment in exosomes over a 24-h period. However, cortisol treatment significantly reduced the expression of Hsp70 in exosomes released from either unstressed or heat-shocked hepatocytes. This cortisol-mediated suppression was not specific to Hsp70 as beta-actin expression was also reduced in exosomes released from hepatocytes treated with the steroid. Our results suggest that circulating Hsp70 is released from target tissues via exosomes, and their release is modulated by stress and cortisol. Overall, we propose a novel role for extracellular vesicular transport of Hsp70 in the organismal stress response.

Vitamin D deficiency and sufficiency among Canadian children residing at high latitude following the revision of the RDA of vitamin D intake in 2010.

Recently, countries at high latitudes have updated their vitamin D recommendations to ensure adequate intake for the musculoskeletal health of their respective populations. In 2010, the dietary guidelines for vitamin D for Canadians and Americans aged 1–70 years increased from 5 μg/d to 15 μg/d, whereas in 2016 for citizens of the UK aged ≥4 years 10 μg/d is recommended. The vitamin D status of Canadian children following the revised dietary guidelines is unknown. Therefore, this study aimed to assess the prevalence and determinants of vitamin D deficiency and sufficiency among Canadian children. For this study, we assumed serum 25-hydroxy vitamin D (25(OH)D) concentrations <30 nmol/l as ‘deficient’ and ≥50 nmol/l as ‘sufficient’. Data from children aged 3–18 years (n 2270) who participated in the 2012/2013 Canadian Health Measures Survey were analysed. Of all children, 5·6% were vitamin D deficient and 71% were vitamin D sufficient. Children who consumed vitamin D-fortified milk daily (77 %) were more likely to be sufficient than those who consumed it less frequently (OR 2·4; 95% CI 1·7, 3·3). The 9% of children who reported taking vitamin D-containing supplements in the previous month had higher 25(OH)D concentrations (β 5·9 nmol/l; 95% CI 1·3, 12·1 nmol/l) relative to those who did not. Children who were older, obese, of non-white ethnicity and from low-income households were less likely to be vitamin D sufficient. To improve vitamin D status, consumption of vitamin D-rich foods should be promoted, and fortification of more food items or formal recommendations for vitamin D supplementation should be considered.

Healthy lifestyle behaviours are positively and independently associated with academic achievement: An analysis of self-reported data from a nationally representative sample of Canadian early adolescents

Introduction The lifestyle behaviours of early adolescents, including diet, physical activity, sleep, and screen usage, are well established contributors to health. These behaviours have also been shown to be associated with academic achievement. Poor academic achievement can additionally contribute to poorer health over the lifespan. This study aims to characterize the associations between health behaviours and self-reported academic achievement. Methods Data from the 2014 Canadian Health Behaviour in School-Aged Children Study (n = 28,608, ages 11–15) were analyzed. Students provided self-report of academic achievement, diet, physical activity, sleep duration, recreational screen time usage, height, weight, and socioeconomic status. Multi-level logistic regression was used to assess the relationship of lifestyle behaviours and body weight status with academic achievement while considering sex, age, and socioeconomic status as potential confounders. Results All health behaviours exhibited independent associations with academic achievement. Frequent consumption of vegetables and fruits, breakfast and dinner with family and regular physical activity were positively associated with higher levels of academic achievement, while frequent consumption of junk food, not meeting sleep recommendations, and overweight and obesity were negatively associated with high academic achievement. Conclusions The present findings demonstrate that lifestyle behaviours are associated with academic achievement, potentially identifying these lifestyle behaviours as effective targets to improve academic achievement in early adolescents. These findings also justify investments in school-based health promotion initiatives.