Rapid and Nongenomic Glucocorticoid Signaling in Rainbow Trout
Dindia, Laura Alexandria
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Corticosteroids are key regulatory hormones involved in many aspects of physiology and have long been known to exert rapid and delayed effects. The delayed corticosteroid effects are mediated by transcriptional events downstream of glucocorticoid receptor activation. Conversely, the rapid effects are mediated independently of transcriptional regulation and are thought to involve non-classical steroid receptors and signaling pathways. Lately, research has begun to focus on delineating the rapid and nongenomic actions of glucocorticoids but most of these studies have been on mammalian models. Cortisol, the primary corticosteroid in teleosts, is an established genomic regulator of the physiological response to stress, but very little is known about either the rapid effects or their mechanisms of action in non-mammalian vertebrates. Additionally, nongenomic glucocorticoid action in the liver is poorly characterized in all animal species despite the importance of this organ in regulating glucocorticoid-mediated physiological adjustments during stress adaptation. The primary objective of this thesis is to investigate the rapid glucocorticoid effects, and their mode of action, associated with stressor-induced corticosteroid elevation in fish liver. The overriding hypothesis is that rapid effects of cortisol on acute stress adaptation involve changes to liver membrane order and rapid modulation of stress signaling pathways in rainbow trout (Oncorhynchus mykiss), a well studied teleost model. This hypothesis was tested by examining rapid plasma membrane and intracellular responses following stressor-induced cortisol elevations in vivo, as well as to cortisol treatment in vitro using liver plasma membrane, tissue slices, hepatocytes in suspension, or primary culture of hepatocytes. Steroid hormones are lipophilic molecules and freely incorporate into the plasma membrane. Through noncovalent interactions (hydrogen bonds and Van der Waal forces), glucocorticoids can potentially alter physical properties of the plasma membrane, thus leading to intracellular responses. The effect of stressor-induced cortisol elevations on physical changes to the hepatic plasma membrane was investigated by measuring the microviscosity of the plasma membrane. Plasma membrane fluidity (inverse of microviscosity) is an important determinant of transmembrane protein function, and changes to lipid order can transmit extracellular signals by activating membrane-associated signaling pathways. Fluidity of purified liver plasma membranes was monitored using steady-state fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene, a well characterized membrane probe. In addition to measuring lipid dynamics, the effect of cortisol on plasma membrane structure and surface properties were also investigated using atomic force microscopy. The effect of cortisol on the activation of key stress signaling pathways, including protein kinase (PKA), protein kinase (PKC), Akt, and mitogen-activated protein kinase (MAPK), was tested in fish liver. Also, as acute stress adaptation is regulated by an integrative hormonal response involving catecholamines (primarily epinephrine), the rapid effect of cortisol action on adrenergic signaling in the liver was evaluated in vitro. Finally, an attempt was made to identify cortisol-binding plasma membrane protein, as glucocorticoids are also thought to mediate rapid effects through a novel membrane glucocorticoid receptor. The results demonstrate for the first time that stressor exposure significantly increases liver plasma membrane fluidity (decreased microviscosity). A role for cortisol in mediating stressor-induced fluidization was confirmed in vitro, as physiological stress levels of this steroid (≥100 ng/ml) significantly increased liver plasma membrane fluidity. In addition to increasing lipid fluidity, acute stress and cortisol treatment altered membrane topography, including changes to membrane microdomains. The stressor-induced cortisol elevation also rapidly modulated major signaling cascades in rainbow trout liver, including PKA, PKC, and ERK1/2 MAPKs. A role for cortisol in the activation of these kinase pathways was confirmed in vitro. Specifically, cortisol rapidly and transiently increases cyclic AMP (cAMP) accumulation and induces the phosphorylation of PKA substrate proteins, including cAMP response element-binding (CREB) protein. In addition to activating PKA signaling, cortisol rapidly induced phosphorylation of PKC and Akt substrate proteins, while stimulating p38 MAPK dephosphorylation in vitro. Moreover, rapid cortisol signaling may stimulate metabolite oxidation in order to maintain the energy balance within liver tissue as cortisol acutely depleted key liver metabolites (including liver glucose), suggesting enhanced turnover without impacting the steady-state adenylate energy charge ratio (measure of the energy status of the cell). Also, rapid effects of cortisol alter the hormonal responsiveness of hepatocytes to adrenergic stimulation, including suppression of epinephrine-stimulated cAMP-CREB activation and glucose production. Preliminary results point to a plasma membrane protein that specifically binds cortisol in trout liver, but this remains to be characterized. Also, in addition the membrane-mediated response, mifepristone, a glucocorticoid receptor (GR)-antagonist, blocked some rapid cortisol effects suggesting the possible involvement of GR signaling pathway. Until now, cortisol has been primarily thought to play a role in the long-term recovery process to acute stress by enhancing plasma glucose levels through the upregulation of liver gluconeogenic capacity. The results presented in this thesis provide evidence for a novel role for rapid cortisol action on the acute metabolic adjustments that support liver function immediately following acute stressor exposure. Particularly, the results lead to the proposal that acute cortisol action stabilizes the energy status of the cell by maintaining ATP levels through increased metabolite turnover, suggesting enhanced metabolic activity of the liver immediately following acute stressor exposure. While the mechanism is unclear, plasma membrane alterations in response to cortisol intercalation may facilitate rapid steroid signaling either through mechanotransduction or by altering activity of plasma membrane proteins. The structural changes to the plasma membrane in response to acute stressor exposure and/or cortisol treatment highlight a novel membrane-mediated mechanism of rapid stress adaptation in hepatic tissue.