Abstract
Glucocorticoids (GCs) circulate in the plasma bound to corticosteroid-binding globulin (CBG). Plasma CBG may limit access of glucocorticoids to tissues (acting as a sponge: the free hormone hypothesis), or may solely serve as a transport molecule, releasing GCs to tissues as the plasma moves through capillaries (the total hormone hypothesis). Both biomedical (focused on human health) and comparative (focused on ecological and evolutionary relevance) studies have worked to incorporate CBG in glucocorticoid physiology, and to understand whether free or total hormone is the biologically active plasma fraction. The biomedical field, however, has been well ahead of the comparative physiologists, and have produced results that can inform comparative research when considering the import of total vs. free plasma hormone. In fact, biomedical studies have made impressive strides regarding the function of CBG in tissues as well as plasma; we, however, focus solely on the plasma functions in this review as this is the primary area of disagreement amongst comparative physiologists. Here we present 5 sets of biomedical studies across genomics, pharmacology, cell culture, whole animal research, and human medicine that strongly support a role for CBG limiting hormone access to tissue. We also discuss three areas of concern across comparative researchers. In contrast to former publications, we are not suggesting that all comparative studies in glucocorticoid physiology must measure CBG, or that only free corticosterone levels are valid. However, we propose that comparative physiologists be aware of biomedical results as they investigate glucocorticoids and interpret how total hormone may or may not impact behavior and physiology of free-living vertebrates.
| Original language | English |
|---|---|
| Article number | 110857 |
| Journal | Molecular and Cellular Endocrinology |
| Volume | 514 |
| DOIs | |
| State | Published - Aug 20 2020 |
Funding
These data demonstrate that changes in CBG can alter total hormone levels. Does this necessarily support the free hormone hypothesis? Or, if CBG is just a transport molecule, not restricting hormone access to tissues, would changes in total hormone occur with increases in CBG? It is unlikely. This would only occur if GC levels were typically higher than CBG capacity to bind GCs. GCs are lipophilic, and so don't dissolve in plasma well. With more CBG, however, more GCs could be carried in the blood, and so total hormone levels could be higher. In the vast majority of studies measuring total hormone and CBG capacity, however, CBG capacity is already large enough to hold even stress-induced levels of hormone in the blood (Charlier et al., 2009; Li et al., 2017). If CBG capacity is always higher than hormone titers, then a change in CBG capacity will only affect free hormone levels (under the law of mass action), not total hormone levels. Under the free hormone hypothesis, an increase in CBG will a) reduce the amount of free hormone entering tissues, b) thereby releasing negative feedback on the HPA axis, and c) increasing total hormone secretion. It is important to note that these arguments are all based on the plasma role of CBG. As mentioned in the introduction, there is strong evidence for extra-hepatic expression of CBG that could function to limit or deliver CORT directly to cells. One example that may affect our outcomes here: CBG present in the pituitary can buffer CORT from binding intracellular receptors, weakening negative feedback on further CORT secretion (Berdusco et al., 1995). If SNPs 1 and 2 explained here also effect tissue-specific expression of CBG, that could separately alter total hormone secretion. This cannot be ruled out as a major driver of plasma total hormone levels; however, our predictions are supported by the functional studies of CBG in the plasma of subjects in the Bolton study, supporting the role of CBG in regulating total hormone levels by its actions in the plasma.Summary of the genomic study: These data support the free hormone hypothesis at a genetic level, suggesting that the primary genetic determinant of total hormone levels is CBG capacity and affinity. Other target gene studies have found as many as 15 human SERPINA6 polymorphisms that have been characterized with defects in the production or steroid-binding activity of CBG (Simard et al., 2015), and many have demonstrated effects on human health (Meyer et al., 2016; Perogamvros et al., 2011, see below). Hence, there is widespread genetic evidence for CBG regulating GC physiology.Evidence presented so far includes genomic and pharmacological evidence supporting the free hormone hypothesis. However, neither example evaluates glucocorticoid action as CBG levels change. If CBG regulates GC access to tissues, then alterations in CBG level should alter GC activity at the cellular level. Perogamvros et al. (2011) tested whether altering CBG capacity in cell culture media could alter glucocorticoid receptor (GR) activation. They evaluated ‘glucocorticoid bioactivity’ by bathing HeLa cells (transfected with human GR-alpha and the MMTV-Luc reporter gene) in human serum that had been stripped of endogenous hormone and spiked with cortisol. Cells were bathed in 10, 20, 50, or 100% serum, each spiked with the same amount of cortisol. Hence, the same amount of total cortisol was available outside the cells, but CBG capacity varied by 10 fold. Perogamvros measured free cortisol levels in both the 100% and 10% sera, demonstrating a much lower level of free hormone in the 100% serum (Fig. 4, light green bars). The total hormone hypothesis predicts that altering CBG capacity would have no effect on GC entrance into cells and therefore GR activation. The free hormone and reservoir hypotheses predict that increased CBG capacity would limit hormone access to cells and reduce GR activation. When human serum was spiked with approximately 300 nM cortisol, more concentrated serum (with greater CBG capacity) had lower free CORT (Fig. 4, light green bars) and lower glucocorticoid receptor activation (black bars). These data suggest that increasing CBG capacity decreases glucocorticoid bioactivity, supporting the free hormone hypothesis. There is, though, a complication here. Throughout this review we have focused on the plasma effects of CBG, setting tissue-specific actions of CBG and CORT-bound CBG aside. There is strong evidence that myriad tissues have CBG present in the extra-cellular fluid or inside the cells, or CBG receptors present in cell membranes (see introduction). There is no evidence for HeLa cells having membrane CBG receptors, but if they do, then conclusions drawn from this study may be more complex.Here we presented 5 different sets of biomedical studies that clearly support the free hormone hypothesis and by extension, the reservoir hypothesis. A). Genomic analysis of over 12,000 human subjects identified only 3 SNPs (of the 2.5 million evaluated) that explain variance in total morning cortisol levels; two are from the CBG gene, and the third produces a protein that protects CBG from cleavage. If CBG only transported, but did not limit hormone access to tissues, changes in CBG affinity and capacity would not alter total cortisol levels. However, if CBG limits access to tissues, alterations in CBG will change how much cortisol can return to tissues to regulate cortisol production, altering total cortisol levels. B) Pharmacological evidence suggests a strong role for binding globulin capacity in regulating hormone clearance rates. Across hormones, hormone binding capacity (the % hormone bound to binding globulin in plasma) closely predicts clearance rate of that hormone. A more detailed study within subjects shows that CBG capacity predicts cortisol clearance rates. If only free hormone can enter tissues to be metabolized and cleared, then binding globulins should regulate clearance rates. C) Experiments using cell culture altered the amount of CBG in the culture media, while keeping cortisol levels constant across treatments. Increased CBG in the media decreased activation of the glucocorticoid receptor, supporting a limiting role for CBG in cortisol access to tissues. D) Microdyalisis studies in rats demonstrate that tissue corticosterone levels mirror free, but not total hormone levels in the plasma through a forced swim test. And finally, E) Human mutations that reduce affinity or eliminate CBG entirely are associated with illness; while mortality rates are predicted by free cortisol but not total, especially in sepsis and septic shock.The authors would like to thank MPG Ranch (CWB) and The American Association of American Women (JLM) for support during the writing of this manuscript, and the thoughtful input of 2 reviewers.
UN SDGs
This output contributes to the following UN Sustainable Development Goals (SDGs)
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SDG 3 Good Health and Well-being
Keywords
- CBG
- Corticosterone
- Cortisol
- Glucocorticoids
- Transcortin
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