ROLE OF CORTICOSTEROID BINDING GLOBULIN IN EMOTIONAL REACTIVITY SEX DIFFERENCES IN MICE

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HPA axis and the glucocorticoids

The HPA axis is the major neuroendocrine system in mammals and, as previously mentioned, its activation culminates in the secretion of glucocorticoids from the adrenal glands. This axis is highly conserved through evolution, with its components being present in early vertebrates. Glucocorticoid hormones are vital and regulate many physiological functions, including growth, glucose, fat, and protein metabolism. They also exert anti-inflammatory and immunosuppressive actions, and can affect mood and cognitive functions (Spiga et al., 2014).

Regulation of the HPA axis

Several endogenous and exogenous factors, such as stress, light, food, sleep, exercise and inflammation can have an impact on the activity of the HPA axis. Exercise represents a physical stress that challenges homeostasis and, therefore, activates the HPA axis, leading to an elevation of cortisol. However, sustained physical conditioning in highly trained athletes is associated with a decreased HPA response to exercise. Interestingly, these highly trained athletes also exhibit a mild but chronic hypercortisolism at basal conditions, which may be an adaptive change to chronic exercise (Mastorakos et al., 2005). Assuming that the stress response is a neuroendocrine mechanism that occurs to prepare the individual to perform a physical action, then physical activity should be the natural means to prevent the consequences of stress. Indeed, accumulating evidence corroborate the beneficial effects of regular exercise in preventing or ameliorating the metabolic and psychological consequences of chronic stress. These benefits are thought to derive from the reduction in the sensitivity to stress, and also from peripheral actions influencing metabolic functions (Tsatsoulis and Fountoulakis, 2006).
The activity of the HPA axis may also impact and be impacted by diet. For example, several studies have used removal of endogenous corticosteroids by adrenalectomy to show that, in the absence of glucocorticoids, feeding and body weight gain are reduced, at the same time that sensitivity to leptin and insulin are increased. Systemic – but not central –administration of glucocorticoids is able to reverse the feeding effects. Interestingly, sucrose ingestion is also capable of restoring food intake and many metabolic deficits following adrenalectomy. Sucrose and lard ingestion have also been shown to decrease CRH messenger ribonucleic acid (mRNA) in the central amygdala, suggesting that ingestion of palatable foods reduces the activity in the central stress response network, perhaps reducing the feeling of stressors (Green et al., 1992, Chavez et al., 1997, Zakrzewska et al., 1997, la Fleur, 2006). Negative energy balance can also be seen as a stressor, threatening the physiological homeostasis. In lean animals and humans, this negative balance has been shown to consistently activate the HPA axis, with emerging evidence of similar changes in overweight and obese people during lifestyle interventions for weight loss (Sainsbury and Zhang, 2012, Seimon et al., 2013).
For being secreted in daily cycles and act on widespread targets throughout the organism, glucocorticoid hormones are obvious candidates to mediate the temporal communication between the suprachiasmatic nucleus (SCN) of the hypothalamus central clock and the rest of the brain and body, regulating circadian rhythms. It has been suggested that, under stable lighting conditions, circulating glucocorticoids reinforce resistance of the circadian timing system to variations of the photoperiod. This would explain why abrupt surges of glucocorticoid concentrations in blood during stress exposure do not interfere in the organism’s circadian rhythms (Sage et al., 2004). Despite the fact that lights on during nighttime may be interpreted as a stressor, glucocorticoids are not critical for the manifestation of changes in metabolism associated with nocturnal illumination. Nighttime light exposure has been inconsistently reported to elevate, reduce, or not affect glucocorticoid secretion, both in animal and human studies (Fonken and Nelson, 2014).
Sleep, in particular deep sleep, has an inhibitory influence on the HPA axis, whereas activation of the HPA axis or administration of glucocorticoids can lead to arousal and sleeplessness. In addition, induced sleep disruption is associated with significant increases of plasma cortisol levels. Furthermore, mean 24-hour plasma cortisol levels are significantly higher in subjects with a shorter total sleep time than those with a longer total sleep time, and insomnia – the most common sleep disorder – is associated with a 24-hour increase of ACTH and cortisol secretion. Sleep deprivation represents a stress to the organism and should be associated with activation of the stress system. However, several studies that have assessed the effects of one night sleep deprivation on the HPA axis found that cortisol secretion either is not affected or is minimally affected following prolonged wakefulness (Moldofsky et al., 1989, Spath-Schwalbe et al., 1991, Steiger and Holsboer, 1997, Brun et al., 1998, Vgontzas and Chrousos, 2002).

Negative feedback regulation of the HPA axis

The HPA axis is governed by a closed-loop negative feedback system typical of most neuroendocrine axes (Figure 5). Glucocorticoid-dependent negative feedback control is essential for the termination of the stress response and reduces the risk of deleterious high amplitude oscillations in circulating levels of glucocorticoids. Normal HPA function is dependent on a dose and duration dependent glucocorticoid-mediated negative feedback. Negative feedback can inhibit the axis by acting at the level of the CRH and AVP neurons of the PVN, through corticotrophs of the anterior pituitary, or indirectly through brain regions containing glucocorticoid receptors that project to the PVN. The influence of corticosteroids on negative feedback depends on the type of ligand (cortisol, corticosterone, synthetic glucocorticoids…) and on the site of action.
Negative feedback of the HPA axis occurs in several time domains. Fast feedback can occur within seconds to minutes, does not depend on protein synthesis and is mediated at the cell membrane level. Evidence suggests that this fast mechanism involve the endocannabinoid system. The delayed feedback, on the other hand, occurs in the minutes to hours time frame and is driven by changes in gene expression through classical actions of glucocorticoid receptors (GR and MR) (Hill and Tasker, 2012, Handa and Weiser, 2014).
Since circulating glucocorticoids can bind either GR or MR, both have been implicated in the negative feedback regulation of the HPA axis, where differential sensitivity to a common ligand appears to allow selective actions. MR has a particularly high affinity for endogenous glucocorticoids and these receptors are predominantly bound under baseline levels of corticosterone. Evidence suggests that MR receptors – particularly those located in the hippocampus – regulate HPA axis activity during the non-stressed state. On the other hand, the relatively lower affinity of GR for endogenous glucocorticoids is thought to direct its actions toward negative feedback following stressors. The GR appears to be largely unoccupied during basal glucocorticoid conditions, but becomes rapidly occupied when glucocorticoid levels increase following stress. This way, GR activation allows the return of HPA activity to baseline following high amplitude peaks of corticosteroids. Similarly to MR, the negative feedback effects of GR on HPA axis following a stressor seems to be mediated by receptors located in the hippocampus, despite the also high expression of this receptor in the PVN and in the anterior pituitary gland. Thus, the ratio of MR to GR may be important for regulating HPA reactivity as well as stress related behaviors (Handa and Weiser, 2014).
Given the traditional view that glucocorticoids work by changing gene transcription, it is surprising to how fast the negative feedback can occur. Powerful glucocorticoid inhibition of the HPA axis occurs within minutes, a time-frame far too fast to be mediated by genomic effect. Thus, rapid feedback must be mediated by non-genomic actions, perhaps working at or near the cell membrane. The mechanism underlying fast feedback remained enigmatic until only recently, when electrophysiology studies suggested that glucocorticoids bind membrane receptors on PVN CRH neurons, eliciting an intracellular cascade that mobilizes the synthesis of endocannabinoids. Endocannabinoid release then causes presynaptic inhibition of glutamate release, which reduces the neural activity of parvocellular neurons (Di et al., 2003). Interestingly, despite these non-genomic effects being proposed to be mediated by membrane-bound receptors, they have recently been shown to still be dependent on the nuclear GR on a study using conditional GR knockout mice with genetic deletion of GR in the PVN and supraoptic nucleus. Nuclear GR would therefore be responsible for transducing the rapid steroid response at the membrane, or be a critical component in the signaling cascade, or regulate a critical component of the signaling cascade of a distinct membrane receptor (Jacobson and Sapolsky, 1991, Herman et al., 2012, Nahar et al., 2015).

Glucocorticoid receptors and genomic responses

It is widely accepted that glucocorticoids exert most of their effects genomically. According to the classic genomic theory of action, glucocorticoids bind to glucocorticoid receptors (MR or GR) located in the cytoplasm of cells from different target tissues. GR and MR differ in their affinity for glucocorticoids. The affinity of the endogenous hormones corticosterone and cortisol for MR is approximately 10-fold higher than that for GR. Therefore, variations in circulating levels of glucocorticoids lead to shifts in the balance between MR and GR occupation, and thus activity. The high-affinity MR is activated at very low concentrations of glucocorticoids, whereas the lower affinity GR is only activated upon high concentrations of glucocorticoids. In basal physiological states, when glucocorticoid concentrations are low, MR is activated and localized in the nucleus, whereas GR remains latent in the cytoplasm. Once glucocorticoid concentrations increase above a threshold level, for example during the circadian peak or following stress exposure, GR translocates into the nucleus where it exerts its genomic effects (Spiga et al., 2014).
MR and GR belong to the superfamily of nuclear receptors. When inactive, they reside in the cytoplasmic compartment, stabilized by chaperone molecules. Upon glucocorticoid binding, the chaperone-receptor complex dissociates and enables the ligand-receptor complex to translocate to the nucleus, where it modulates the transcriptional activity of glucocorticoids responsive genes. This positive or negative modulation of the expression of glucocorticoids target genes happens either through the binding of the complex to specific sequences – glucocorticoid-response elements (GREs) – in the promoter region of target genes, or through protein-protein interactions with other transcription factors, such as nuclear factor-кB (NF-кB), activator protein-1 (AP-1), and several signal transducers and activators of transcription (STATs) (Jiang et al., 2014, Spiga et al., 2014). MR and GR are differently distributed throughout the body. While GR is ubiquitously expressed in the periphery and in the brain, the distribution of MR is more restricted to specific organs such as the kidney and the heart. In the brain, the distribution of these receptors is also not uniform. GR is almost ubiquitously expressed in neurons and glial cells, with particularly high density in the hippocampus and in parvocellular neurons of the PVN. In contrast, MR localization is restricted to the hippocampus, PFC and amygdala, structures highly involved in learning, memory, and emotional behavior. Both receptors are abundantly expressed in areas regulating the HPA axis function, including a number of limbic structures that regulate the activity of the PVN, such as the hippocampus and amygdala, as well as in the anterior pituitary. In addition to the overlap in GR and MR expression within hippocampus and cortex, GR and MR can be co-expressed within individual cells. Interestingly, brain regions or cells that express both receptor types are capable of mediating varied responses to the endogenous glucocorticoid, according to the occupancy level of each type of receptor (van Eekelen et al., 1991, Handa and Weiser, 2014, Spiga et al., 2014).

Non-genomic effects of glucocorticoids

One of the most important actions of glucocorticoids is to enhance the response to stress and to protect the organism from specific challenges to homeostasis. Almost any type of stress, whether physical or psychogenic, causes a rapid and marked increase in ACTH secretion by the anterior pituitary gland, followed within minutes by greatly increased adrenocortical secretion of glucocorticoids. Even though it is well known that glucocorticoid secretion increases significantly in stressful situations, and that this enhances tolerance to stress, it is believed traditionally that glucocorticoids exert these effects genomically. Non-genomic effects of glucocorticoids are characterized as short latency and insensitive to inhibitors of deoxyribonucleic acid (DNA) transcription and protein synthesis compared with genomic effects, which require at least 30 min and up to several hours or days to take effect. Thus, we can infer that the non-genomic effects of glucocorticoids play an important role in the early phase of acute stress. In fact, non-genomic effects of glucocorticoids have already been described for a series or processes, such as the inhibition of inflammatory factors and the inflammatory response, the maintenance of blood pressure by potentiating the effect of catecholamines and the elevation of blood glucose concentrations. All these phenomena were described to take place in less than 20 min after the administration of glucocorticoids (Jiang et al., 2014).
Although some studies indicate that nuclear MR and GR receptors can initiate second messenger signaling or interact with other cellular signaling components, there is growing body of evidence to suggest that these rapid actions are mediated by specific receptors localized at the plasma membrane. Membrane-impermeable glucocorticoid conjugates are able to trigger these rapid actions, which directly supports the membrane-initiated signaling. These effects seem to be mediated by G protein coupled membrane-associated receptors, with a pharmacological profile different from the well-known intracellular receptors. The inhibitory activity of glucocorticoids on the HPA axis is thought to be mediated by these membrane receptors, given the rapidity of this effect at both the level of the pituitary and the brain (Spiga et al., 2014).
One interesting example of an effect mediated by membrane receptors is the rapid and non-genomic effects of acute stress on memory retrieval. An acute stress (electric footshock) induces a corticosterone rise in the dorsal hippocampus and memory retrieval impairment in a spontaneous delayed alternation task. Bilateral injections of either corticosterone or a corticosterone-bovine serum albumin (BSA) complex (impermeable to the plasma membrane) in the dorsal hippocampus before memory retrieval produced impairments similar to those resulting from acute stress. Furthermore, bilateral intra-hippocampal injection of RU-28318 (an MR antagonist) but not of RU-38486 (a GR antagonist) totally blocked the memory retrieval deficit induced by the corticosterone–BSA complex administration. This demonstrates that the membrane receptors mediating the rapid and non-genomic effects of acute stress on memory retrieval are of MR type (Dorey et al., 2011).

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Circadian and ultradian influences on the HPA axis

The term circadian comes from the Latin circa diem, meaning “around a day”. Circadian rhythms refer to biological oscillations within a period of approximately 24 hours. These rhythms are highly conserved among living organisms, and are characteristic of a number of biochemical, physiological, endocrine, and behavioral functions, allowing the organism to anticipate and prepare for predictable environmental changes, such as food availability, predator risk, and the likelihood of reproductive success. The result is an optimization of energy expenditure by an optimal coordination of physiological processes (Kalsbeek et al., 2012, Spiga et al., 2014).
In mammals, circadian rhythms are generated and synchronized by a central clock in the SCN of the ventral hypothalamus. The activity of the SCN is entrained by the light input received from the retina, and this allows the organisms’ physiology to be entrained to solar time. The molecular mechanism regulating the circadian activity of each single cell within the SCN consists of an oscillating transcriptional network of a series of “clock genes”, regulated by a network of positive and negative transcriptional, translational and posttranslational feedback loops, resulting in the rhythmic expression of their protein products. Signaling between the SCN and the periphery involves both hormonal and neuronal mechanisms. Rhythmic activity of the SCN is translated into circadian patterns of gene expression in target tissues, which in turn determine circadian rhythms in the physiological function of that specific organ. In rodents, lesions in the SCN result in a loss of physiological circadian rhythmicity, including body temperature, locomotor activity, and hormone secretion, even when the 24 hours light-dark cycle is maintained, and the circadian rhythm of locomotor activity can be reinstated in arrhythmic animals by transplantation of an intact SCN (Stephan and Zucker, 1972, Abe et al., 1979, Ralph et al., 1990, Buijs et al., 1993). In addition to the central master clock in the SCN, clock genes are also expressed rhythmically in peripheral tissues, including liver, kidney, skeletal muscle, lung, and adrenals. However, although they share the same molecular dynamics of clock genes, only the SCN can independently generate and maintain its own circadian rhythms, while peripheral clocks require SCN-dependent drive to maintain synchronicity with the light-dark cycle (Reppert and Weaver, 2002, Welsh et al., 2004, Levi and Schibler, 2007, Spiga et al., 2014).
The HPA axis operates under a circadian timing mechanism that is both diurnal and ultradian in nature. The circadian rhythm of the HPA axis is primarily regulated by the central pacemaker in the SCN. The SCN regulates the circadian rhythm of glucocorticoids in part by modulating the release of CRH from neurons in the PVN, and in part via the autonomic nervous system, through a multisynaptic neural pathway from the SCN to the adrenal gland. Additionally, there is evidence of an intra-adrenal circadian pacemaker that can regulate glucocorticoid synthesis independently from the SCN (Oster et al., 2006, Son et al., 2008). Evidence for the involvement of adrenal clock genes in circadian glucocorticoid synthesis has been shown in vivo using mice defective in components of the circadian clock system. In fact, studies in clock gene knockout mice have shown that disruption of the circadian clock in these animals is associated with a disruption of the HPA circadian rhythm. The circadian rhythm of clock genes in the adrenals was also shown to be independent of circadian rhythmicity of ACTH, corroborating the important role of clock genes in the adrenals in regulating circadian rhythms of glucocorticoid synthesis (Spiga et al., 2014).

Variability of CBG levels

CBG binds mainly to glucocorticoids, and thus regulates the bioavailability and metabolic clearance of these hormones to their target tissues. The levels of plasma CBG are the result of their production and elimination rates, and every factor influencing either of these processes can potentially influence the plasma levels of this glycoprotein. Other factors may also influence CBG’s activity, by modulating its binding affinity. All these processes may vary normally under physiological conditions, but some variations are associated with pathological states. Age, development, hormonal influences, stress and sex are some of the factors that can influence CBG levels and activity.

Variability among species

Plasma levels of CBG vary from 10 to 500 nM in most vertebrate species. They are exceptionally high in the green iguana (3000 nM) and squirrel (2500 nM), and they are low in New World monkeys – only 1-10% of those in other primates. The affinity is also lower in New World monkeys, resulting in a much higher fraction of unbound plasma cortisol. In the squirrel monkey, the only mammal known that appears to lack CBG, half the cortisol is albumin-bound and half is free (Dunn et al., 1981). A study that compared the CBG binding of cortisol measured by equilibrium dialysis in seven species revealed that CBG maximal capacity (Bmax) was approximately three-times more than the plasma cortisol levels for most species, with cow, dog and ewe exhibiting the lowest and cynomolgus monkey exhibiting the highest values. The free (6 to 14%), CBG-bound (67 to 87%), and albumin-bound (7 to 19%) cortisol fractions are also similar within species. Also in most species, as much as 68%of plasma CBG was found to remain free of cortisol under physiologic conditions (Gayrard et al., 1996).

Physiological variability

The concentration of CBG in the lymph is slightly lower than that in the blood, whereas  concentrations in the synovial fluid and pleural effusion are higher and those in the spinal fluid and amniotic fluid are very low. CBG passes into milk – in human milk the concentration has been reported to be 1-15% that of plasma and in rat milk approximately 25% that of plasma. Because there is little or no CBG or albumin in saliva, salivary cortisol is often used as an index of free cortisol. However, the salivary gland contains HSD11B2, which converts approximately half the cortisol to cortisone, so salivary values are approximately one-half those of free cortisol in plasma (Murphy, 2010).
CBG production is apparently independently regulated in mother and fetus. Whereas fetal corticosteroid levels rise in many species during pregnancy, CBG levels vary considerably with species; in many, the levels fall rapidly postpartum, and in most species the early postpartum fetal CBG levels are lower than adult values. Compared to the adult (700 nM), CBG levels are lower in the human fetus and neonate: approximately 250 nM in late gestation and immediately postpartum. These levels rise to approximately 500 nM by 30 days postpartum, and reach adult levels by several months of age.
The level of CBG in the fetal rat rises from approximately 1.3 nM at 16 days gestation to a maximum of 2 nM at 18 days. Between gestation day 18 and postnatal day 3 these levels fall and reach almost undetectable values, and start rising again at postnatal day 10 until reaching adult values by 6 weeks. Despite the low neonatal values of CBG in rats, hippocampal glucocorticoid receptor occupancy was noted to be similar at all ages, both under basal conditions and following stress. The decline in fetal CBG before birth has been suggested to promote an increase in free corticosterone levels necessary for lung maturation (Brien, 1981, Viau et al., 1996, Murphy, 2010). CBG levels are higher in younger adults than in older adults. Furthermore, CBG levels do not differ greatly among young men and women, but levels are higher in older women compared to older men (Kudielka et al., 2004). Slight changes in CBG concentrations have been reported to occur with diet, with a high carbohydrate diet resulting in a fall in CBG concentrations, and low fat feeding resulting in higher plasma CBG concentrations and lower free cortisol values (Garrel, 1996, Murphy, 2010).

Table of contents :

1Stress
1.1 The concept of stress
1.1.1 The stress response as a complex system
1.1.2 Stress response: from adaptation to disease
1.2 HPA axis and the glucocorticoids
1.2.1 Regulation of the HPA axis
1.2.2 Negative feedback regulation of the HPA axis
1.2.3 Glucocorticoid synthesis
1.2.4 Glucocorticoid Metabolism
1.2.5 Glucocorticoid receptors and genomic responses
1.2.6 Non-genomic effects of glucocorticoids
1.2.7 Circadian and ultradian influences on the HPA axis
1.2.7.1 Effects of glucocorticoid pulsatility in the brain
1.2.8 The actions of glucocorticoids
1.2.8.1 Actions on growth and development
1.2.8.2 Actions on the immune and inflammatory responses
1.2.8.3 Actions on the metabolism
1.2.8.3.1 The impact of chronic stress on body weight
1.2.8.4 Actions on the central nervous system
1.2.8.4.1 Glucocorticoids and mood regulation
1.2.8.4.2 Glucocorticoids and memory
1.2.9 Variability in the HPA axis responses
1.2.9.1 Sex and gender
1.2.9.2 Genetic factors
1.2.10 Animal models of HPA axis alterations
1.2.10.1 CRH and CHR receptor
1.2.10.2 AVP and AVP receptor
1.2.10.3 ACTH receptor
1.2.10.4 Mineralocorticoid and glucocorticoid receptors
1.2.10.5 11-β-hydroxysteroid dehydrogenases
1.3 Corticosteroid binding globulin (CBG) or transcortin
1.3.1 Structure
1.3.2 Biosynthesis
1.3.3 Glycosylation
1.3.4 Clearance
1.3.5 Binding specificity
1.3.6 Binding dynamics
1.3.7 Interaction with cellular membranes
1.3.8 Variability of CBG levels
1.3.8.1 Variability among species
1.3.8.2 Physiological variability
1.3.8.3 Sexual dimorphism
1.3.8.4 Circadian variability
1.3.8.5 Changes in response to stress
1.3.8.6 CBG alterations during disease
1.3.8.7 Genetic variants and polymorphisms
1.3.8.7.1 In animal models
1.3.8.7.2 In humans
1.3.8.7.3 The Cbg ko mouse: a model of CBG dysfunction
OBJECTIVES
STUDY I – ROLE OF CORTICOSTEROID BINDING GLOBULIN IN EMOTIONAL REACTIVITY SEX DIFFERENCES IN MICE
Introduction
Article
Complementary studies
STUDY II – CHRONIC STRESS DOES NOT FURTHER EXACERBATE THE ABNORMAL PSYCHONEUROENDOCRINE PHENOTYPE OF CBG-DEFICIENT MALE MICE
Introduction
Article
Complementary studies
STUDY III – CBG DEFICIENCY SPECIFICALLY IMPAIRS HIPPOCAMPUS-DEPENDENT MEMORY CONSOLIDATION IN MALE MICE
Article (under preparation)
Complementary studies
STUDY IV – BEHAVIORAL AND METABOLIC CONSEQUENCES OF CBG DEFICIENCY ON MALE MICE SUBMITTED TO HIGH-FAT DIET COMBINED TO CHRONIC MILD STRESS
Introduction
Article (under preparation)
Conclusions
REFERENCES

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