Mediating free glucocorticoid levels in the blood of vertebrates: are corticosteroid-binding proteins always necessary?

Glucocorticoids (GC) are integral to the stress response of vertebrates to environmental challenges. Their impact is immediate and widespread, and prolonged exposure can result in major activational and organizational changes. The vertebrate body has two mechanisms to limit GC impact: first, a rapid negative feedback system to turn off their release, and second, a protein corticosteroid-binding globulin (CBG) to prevent them being free in the blood. Species used in biomedical research have CBG levels that normally bind 9095% of blood GC. This evidence was the basis for the Free Hormone Hypothesis, which posits that only unbound, free hormone is available for use by tissues and is biologically active. High levels of free GC typically occur only under conditions of chronic stress and are associated with major suppression of key body functions. The hypothesis proposes that the primary role of CBG is to render GCs unavailable, thereby preventing tissue exposure. From a field study in southern Ontario on northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels, we found virtually no CBG binding capacity in their plasma, resulting in only about 10% of cortisol being bound throughout the year. However, neither species showed any evidence of being physiologically compromised. This presents a major challenge to the potential consequences of chronically high levels of free GC. To assess the generality of this finding, we carried out a phylogenetic comparison of 91 vertebrate species in which both GC and CBG levels were measured. In 93%, CBG levels were sufficient to bind about 90% of circulating GCs. This evidence is thus concordant with that from biomedical research. However, both flying squirrel species and four species of New World monkeys were extreme outliers. These two groups had the highest GC concentrations, but relatively little binding capacity (10% or less of their GC was bound). An ancestor state reconstruction of the proportion of GCs bound indicated that the flying squirrels and New World monkeys evolved this character state from ancestors that followed the common 90% bound pattern. It also indicated that the original vertebrates the earliest fishes have most of their GC in the free state and little or no steroid-binding protein. We thus demonstrate a dichotomous pattern with respect to CBG: a dominant branch, where high levels of CBG bind most of the GC, applies to the majority of vertebrates; and a secondary branch, where low levels of CBG bind almost none of the GC, applies to a very small subset. For the latter, the critical unknown is how these species mitigate the impact of the high free GC levels and how such a dramatic trait shift could evolve.