Functional Ecology 2013, 27, 107–119
doi: 10.1111/1365-2435.12038
THE ECOLOGY OF STRESS
Mediating free glucocorticoid levels in the blood of
vertebrates: are corticosteroid-binding proteins always
necessary?
Lanna M. Desantis*,1, Brendan Delehanty1, Jason T. Weir2 and Rudy Boonstra1
1
Centre for the Neurobiology of Stress, Department of Biological Sciences, University of Toronto Scarborough, Toronto,
Ontario M1C 1A4, Canada; and 2Department of Biological Sciences, University of Toronto Scarborough, Toronto,
Ontario M1C 1A4, Canada
Summary
1. 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 90–95% 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.
2. 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.
3. 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).
4. 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.
5. 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.
Key-words: chronic stress, corticosteroid-binding globulin, field endocrinology, flying
squirrels, free hormone hypothesis, Glaucomys, glucocorticoid receptors, stress axis, stress
physiology
*Correspondence author. E-mail: [email protected]
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society
108 L. M. Desantis et al.
Introduction
The stress axis (the limbic system [dentate gyrus and hippocampus] combined with the hypothalamic–pituitary–
interrenal axis in fish, amphibians and reptiles and the
hypothalamic–pituitary–adrenal (HPA) axis in birds and
mammals) is one of the key neuroendocrine control mechanisms that mediate the relationship of the organism to its
environment. This axis has two essential functions in vertebrates. First, it is the critical axis involved in the normal
diurnal cycle of waking involving increased locomotion,
exploratory behaviour, increased appetite and food-seeking
behaviour (reviewed in McEwen, Brinton & Sapolsky
1988; Wingfield & Romero 2001). Second, it permits both
short-term responses to environmental stressors (e.g. predator attack, social interaction; Wingfield & Romero 2001)
and long-term evolutionary responses to particular ecological pressures (Wingfield & Sapolsky 2003; Boonstra 2005;
Breuner, Patterson & Hahn 2008; Hau et al. 2010).
Stressors activate a rapid cascade of hormonal responses
that, in the HPA axis, culminates in the release of glucocorticoids (GCs) (corticosterone and/or cortisol, depending
on the species) from the adrenal cortex. GCs are known to
influence the expression of approximately 10% of the genome, and its targets include genes controlling metabolism,
growth, repair, reproduction and the management of
resource allocation (Le et al. 2005). GCs signal the body to
mobilize energy, suppress physiological processes not
required to deal with the stressor and then act to maintain
homoeostasis after the stressor has passed. Under conditions when the stressor is acute, termination of the stress
response is accomplished rapidly by GCs exerting negative
feedback (primarily to the pituitary) to return the body
back to the pre-activation state. Under conditions when the
stressor is chronic, termination of the stress response is
muted, and GCs remain high for a prolonged period of
time, leading to the mobilization of energy at the cost of
energy storage, and to suppression of growth, reproduction,
digestion and the immune and inflammatory responses (for
laboratory evidence, see: Dallman et al. 1992; Sapolsky,
Romero & Munck 2000; for field evidence, see: Boonstra
et al. 1998; Wingfield & Sapolsky 2003). The stress axis is
thus a vital regulator of adaptation in vertebrates.
A key component of this regulation involves corticosteroid-binding globulin (CBG). CBG is a protein produced
by the liver that circulates in the blood and binds GCs
with high affinity and low capacity (Westphal 1983). In
laboratory rodent models and humans, 90–95% of total
GC levels are bound to CBG, and its primary role may be
to keep GCs biologically inert (Rosner 1990; Mendel 1992;
Breuner & Orchinik 2002). Free, unbound GCs are
expected to more readily diffuse into the cell through the
phospholipid plasma membranes, binding to GC receptors
to initiate signal transduction. This expectation is the basis
for the ‘Free Hormone Hypothesis,’ which states that only
hormone that is free and not bound to CBG contributes to
the intracellular concentration of free hormone and results
in biological activity (Mendel 1989, 1992). Under nonstressful conditions, vertebrates should maintain a binding
capacity in the plasma capable of keeping most of the
basal circulating GCs in a bound reservoir. Each CBG
molecule has a single binding site (Westphal 1983), and so
one would predict that the molar concentration of plasma
CBG would be approximately equal to or greater than that
of total plasma GCs at any one time. This is the case in
humans (e.g. Angeli et al. 1977; Gayrard, Alvinerie &
Toutain 1996) and in all laboratory rodent species (e.g.
rats: Taymans et al. 1997; mice: Richard et al. 2010).
There are three additional roles for CBG. First, the
bound fraction of hormone is thought to act as a reservoir,
from which GCs are slowly released as the free fraction is
taken up for use by tissues (Rosner 1990; Breuner & Orchinik 2002), and this was corroborated by Perogamvros et al.
(2011). GCs can be lost through metabolization by the liver
and then excreted in both the urine and the faeces (Taylor
1971; Palme et al. 2005). It is assumed that only free GCs
are metabolized (Palme et al. 2005), and a recent study supports this assumption (Sheriff, Krebs & Boonstra 2010).
Second, CBG aids in the delivery of GCs to site-specific
areas of inflammation, where only the arrival of the bound
complex can be utilized to help control the inflammatory
response (Pemberton et al. 1988; Lin, Muller & Hammond
2010; Perogamvros et al. 2011). Third, the CBG–GC complex may bind to membrane receptors to initiate signalling
cascades for physiological action in some tissues (reviewed
in Hammond 1995; Breuner & Orchinik 2002).
Stress, however, affects CBG levels. Though CBG levels
show little variation in response to short-term acute stress,
longer-term acute stress and chronic stress cause CBG levels to decline (laboratory species: e.g. Schlechte & Hamilton 1987; Armario et al. 1994; Fleshner et al. 1995; wild
species: e.g. Boonstra et al. 1998; Breuner et al. 2006; Delehanty & Boonstra 2009), resulting in an increase in free
GC levels. This, coupled with the fact that chronic stress
also desensitizes the negative feedback mechanism
(described earlier), leads to high and persistent levels of
GCs. Under these conditions, the harmful effects of GCs
are exacerbated.
It is clear from the above that CBG plays a critical role
in most species in modulating free GC levels and limiting
exposure of body tissues to them. From a recent comparative study of squirrel species in southern Ontario (unpublished), we discovered extremely high cortisol levels
coupled with low CBG levels in both northern (Glaucomys
sabrinus) and southern (Glaucomys volans) flying squirrels
(photo: Fig. 1) under conditions when they were not chronically stressed. Their tissues must therefore be continually
exposed to high levels of biologically active cortisol, and
thus, they deviate markedly from the 90% binding convention found in laboratory rodents and humans. From the literature on the HPA axis in vertebrates, we know that there
is variation in GC (Romero 2002) and CBG (Breuner &
Orchinik 2002) levels. However, no one has comprehensively assessed the extent of this variation. Thus, how
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
Mediating free glucocorticoid in vertebrates
exceptional is the character state found in flying squirrels
and how extensive is the variation among vertebrates?
To address these questions, we first analysed our seasonal data for levels of endogenous plasma cortisol (total
and free) and CBG binding capacity in these flying squirrels and present them here. To calculate free cortisol levels,
we needed to know the association constant (Ka) between
cortisol and CBG, and we determined this by measuring its
inverse, the dissociation constant (Kd). We then carried out
a comparative phylogenetic analysis on all total GC levels
and CBG binding capacities in vertebrates available from
the published literature to quantify the variation in these
attributes. This permitted us first, to assess the uniqueness
of the character state found in flying squirrels and speculate
as to how they have persisted through evolutionary time,
and second, to assess the evolutionary history of CBG by
shedding light on where it is absent and on its origins.
Materials and methods
FLYING SQUIRREL PLASMA COLLECTION
Both species of flying squirrels were live-trapped seasonally from
May 2008 to March 2009 in the Kawartha Lakes area of Ontario,
Canada near Mississauga Lake (44°41′N/78°10′W). In this area,
they are sympatric. The site is on the southern edge of the Canadian Shield and contains second growth mixed deciduous forest
dominated by white pine and red oak, with a mixture of white
oak, sugar maple, white and yellow birch, large-toothed aspen and
American beech.
Tomahawk live-traps were set and baited with peanut butter at
dusk and checked 3 h after dark. Each night, captured animals
were transported to a central location immediately adjacent to the
trapping grid for processing. On average, two to three squirrels
were captured per night. Squirrels were anesthetized with isoflurane (Abbott Laboratories, Montreal, QC, Canada), and blood was
then drawn from the sub-orbital sinus (200–500 lL) with heparinized Pasteur pipettes and kept on ice until sampling was complete.
Squirrels were therefore held in traps for a minimum of 1 h and a
maximum of 5 h prior to blood sampling. Thus, all blood samples
were from squirrels stressed by capture and handling. Plasma was
removed after centrifugation, and samples were stored at 80 °C
109
until processing. All procedures follow Canadian Council of Animal Care guidelines and were approved under a University of
Toronto Animal Use Protocol (# 20007021).
CORTISOL AND CBG ASSAYS
Total plasma cortisol was measured using a commercially available radioimmunoassay (Clinical Assays GammaCoat Cortisol
125
I RIA Kit; DiaSorin, Stillwater, MN, USA). This kit was validated for parallelism on flying squirrel plasma. Tests for differences between slopes on log-transformed data showed that serially
diluted plasma curves for both species were parallel to the assay
standard curve (southern flying squirrels: F1,11 = 040, P = 054;
northern flying squirrels: F1,11 = 075, P = 041). The intra- and
inter-assay coefficients of variation (CV) were 53% and 103%,
respectively. Maximum corticosteroid-binding capacity (MCBC),
a measure of systemically available CBG, was measured using the
method presented in Delehanty & Boonstra (2009). The intra- and
inter-assay CV were 86% and 145%, respectively. Binding capacities throughout the results section will be referred to as MCBC.
CALCULATION OF CBG BINDING COEFFICIENTS AND
FREE CORTISOL LEVELS
Free cortisol concentrations were estimated according to the
method of Barsano & Baumann (1989). This estimate of free hormone requires knowledge of the species-specific association constant (Ka), which indicates the strength of the affinity of CBG for
its GC. This binding property is ordinarily determined and
reported as its inverse, the equilibrium dissociation constant (Kd),
which represents the concentration of free hormone at which
bound and free fractions of total hormone concentration are at
equilibrium in the plasma. A low Kd (i.e. high Ka) indicates a
strong affinity of CBG for GC, and a high Kd (i.e. low Ka) indicates a weak affinity. Our method for calculating the Kd was
adapted from the saturation binding procedure of Hammond &
L€
ahteenm€
aki (1983). To compare the CBG binding properties of
flying squirrels to a mammal with more typical binding patterns,
we used plasma from red squirrels (Tamiasciurus hudsonicus) that
had been collected in a previous study (see Boonstra et al. 2008
for details). Red squirrels are a diurnal, arboreal species that are
closely related to flying squirrels and share the same forests.
For clarity, we will define the following terms associated with
protein binding properties that we use throughout the results and
discussion sections: total binding refers to cortisol bound to both
CBG and serum albumin; non-specific binding refers to cortisol
bound only to serum albumin; specific binding refers to cortisol
bound only to CBG; and Bmax equals the maximum specific binding capacity of CBG, as determined in our saturation binding
assays.
ASSEMBLY OF PUBLISHED DATA
Fig. 1. Adult northern flying squirrel (Glaucomys sabrinus) gliding
at night in the Kawartha Lakes region of Ontario, December
2008. Photo: Glenn Abuja.
To assess how MCBC levels varied as a function of total GC levels, we obtained both values for all species from which they were
available in the published literature. Selection criteria were based
on four main components: (i) all data were from animals within
the subphylum Vertebrata with species representing all major vertebrate groups (fish, amphibians, reptiles, birds and mammals); (ii)
only studies measuring CBG levels (MCBC) and/or measuring or
estimating free GC concentrations in plasma or serum in addition
to total concentrations were included; (iii) estimates were included
only for normal, healthy animals (i.e. animals not manipulated by
drugs, affected by disease or by other treatment conditions); and
(iv) all data were from adult (breeding) animals, except for bats
where adult data were pooled with that of juveniles due to lack of
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
110 L. M. Desantis et al.
statistically significant differences among age groups to increase
sample size (M. Timonin & C. Willis, unpublished data). The studies we cite used three different methods to estimate MCBC (see
Table S1, Supporting Information), and this could have introduced
variation. To the best of our knowledge, no study has used each of
them on the same plasma samples to assess their comparability.
However, a number of studies have used different methods on the
same species (i.e. humans and horses), and comparable MCBC values were obtained (Table S1, Supporting Information). This suggests that the error induced by these different methods is minor.
When means for any of the four parameters used in this study
(total, free and% free GC, and MCBC) were presented in figures,
but not in a table or in the text, we extracted data from digitized
figures using the shareware program DataThief III (Tummers
2006). For ease of comparison, all hormone and MCBC/CBG concentrations were converted to ng mL 1 of plasma or serum. Data
meeting the above criteria are included in Table S1 (Supporting
Information) in standard Excel format (Version 11.6, Excel 2004
for Mac, Microsoft, Redmond, WA, USA), and related references
are listed in Appendix S1 (Supporting Information).
COMPARATIVE ANALYSES
Although many species are represented by seasonal data from
more than one point in time, or by more than one study, we chose
only one set of data per species for statistical analysis to avoid
pseudo replication; these are indicated by an asterisk following the
species’ common name in Table S1 (Supporting Information). To
make the data as comparable as possible among species, we chose
data from males in the mid-breeding season whenever available.
When this was not possible, the species was represented by data
from females in mid-breeding season, or by the only values available from studies that did not collect seasonal data or did not distinguish between the sexes. In addition, because the original data
from flying squirrels represent nominal hormone levels due to
trapping and handling (i.e. not true baseline levels), we chose
nominal values from other species whenever possible. For studies
including only true baseline hormone levels, as in most of the
captive and domestic species, or where this information was not
stated, then the only data available were included in our analysis.
Table S1 (Supporting Information) lists data for 96 vertebrate
species, but only 91 species are used for phylogenetic comparison.
Sample sizes for each of the major groups are: fish = 9; amphibians = 2; reptiles = 11; birds = 14; mammals = 55. Four species of
salmonid fish were removed from analyses because the data were
collected from the post-spawning period shortly before they die;
these values do not reflect cortisol and CBG-type binding found
at all other times of the year. Salmon are unique in that they die
from exposure to chronic stress during spawning when free cortisol levels are extremely high due to the breakdown in production
of binding protein (Idler & Freeman 1968). This group is represented with pre-spawning data from semelparous chinook salmon
(Barry et al. 2001), which shows that their stress axis is ‘normal’
up until breeding. Their post-breeding strategy is noteworthy however and is later discussed. Brush-tailed phascogales were also
removed from analyses because their total hormone levels reflect
laboratory and not natural field conditions (Schmidt et al. 2006),
which may not be truly representative of this species since they are
not commonly used as laboratory animals, and since data from
their marsupial relatives are from field studies.
Comparison of residuals from a biologically archetypal
trend line
A scatter plot of MCBC vs. total GC values for 91 species was
constructed. To quantify the degree of variation in these attributes
among vertebrates, a trend line representing the concentration of
CBG required to bind 90% of the total GC available was plotted
using the median Kd value (13 nM) of all those listed in Table S1
(Supporting Information). A proportion bound of 90% was chosen because most laboratory and domestic mammals and birds
show only about 10% free GC at any one time, and these species
represent the benchmark for the free hormone hypothesis. Absolute (i.e. untransformed) residuals from this theoretical, but biologically relevant trend line were calculated and their frequency
distribution plotted. The Kolmogorov–Smirnov test for normality
was performed, and the P-value for this test was calculated using
the Dallal and Wilkinson approximation to Lilliefors’ method.
The degree of skewness and kurtosis was also determined. PRISM 4
for Macintosh (GraphPad Software, Inc., San Diego, CA, USA)
was used to create figures and perform statistical tests.
Ancestor state reconstruction
An ancestor state reconstruction of log MCBC as a proportion
of log total GC was performed in Mesquite (Maddison &
Maddison 2010) using sum of squared changes parsimony algorithm (Maddison 1991). This method uses continuous species
characters to calculate ancestor states where the linear cost
assumption from state x to state y along a given branch is
(x y)2 (Maddison 1991). Total cost across the tree is the sum
of costs across all branches. The reconstruction with the lowest
total cost is the most parsimonious. Tree topology used for
ancestor state reconstruction was fixed based on published
molecular phylogenetic studies (topology at and above level of
order: Alfaro et al. 2009; Campbell & Lapointe 2011; turtles:
Krenz et al. 2005; salmonids: Crespi & Fulton 2004; pinnipeds:
Davis et al. 2004; primates: Fabre, Rodrigues & Douzery 2009;
sciurids: Mercer & Roth 2003; Harrison et al. 2003).
Results
MAXIMUM CORTICOSTEROID BINDING CAPACITY VS.
TOTAL GLUCOCORTICOID LEVELS IN FLYING
SQUIRRELS
Both northern and southern flying squirrels exhibited
exceptionally high levels of total cortisol, with seasonal
averages ranging from 79276 to 201429 ng mL 1, and
low levels of MCBC ranging from 5068 to
13469 ng mL 1 (Table 1). This finding was consistent
between males and females and across all seasons in both
species.
Both flying squirrel species showed similar saturation
binding curves, and we therefore present only the results
for southern flying squirrels (Fig. 2a). There was essentially no specific binding, and thus, total binding was
equivalent to non-specific binding. Without a reliable saturable limit (Bmax), we could not calculate a species-specific
Kd, indicating that it is very high. In contrast, red squirrels
had a typical saturation-binding curve (Fig. 2b), with specific binding achieving a saturable limit, resulting in a Kd
of 288 nM. To estimate free cortisol levels in flying squirrels, we used the red squirrel Kd value. Even with the use
of this conservatively low Kd, the high endogenous cortisol
levels and the low binding capacities resulted in free cortisol estimates of at least 90% of total GC (Table 1).
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
Mediating free glucocorticoid in vertebrates
MAXIMUM CORTICOSTEROID BINDING CAPACITY VS.
TOTAL GLUCOCORTICOID LEVELS AMONG
VERTEBRATES
In our analysis comparing MCBC and total GC levels to
the 90% bound trend line (Fig. 3), the vast majority of
species fall on or close to the line. However, a small number fall well below the line, indicating little binding capacity relative to their total GC levels. The most extreme
cases are the North American flying squirrels and four
New World monkey species from South America. These
species have the highest total GC levels (ranging from
99097 to 226175 ng mL 1; Fig. 3 and see Table S1, Supporting Information for all values), but the lowest MCBC
levels (ranging only from 906 to 12523 ng mL 1). These
two groups also differ markedly from their closest relatives, with both having related species (‘Other Squirrels’
and ‘Old World Primates’) falling on or very close to the
trend line (Fig. 3). Some species of fish also have values
well below the trend line, though most are close to or
above it. Their total GC levels however, are generally
much lower (all <470 ng mL 1). Meadow voles appear at
the opposite end of the spectrum, having total GC levels
(37660 ng mL 1) that are markedly exceeded by very high
binding capacity (168310 ng mL 1).
A plot of residuals from the trend line (Fig. 4) indicates
a distribution that deviates markedly from normality
(D = 03254; P < 00001). The vast majority of species
(93%) have residuals near zero (within 400 units), very
111
few have intermediate negative or positive residuals, and
some have pronounced negative or positive residuals. A
negative skew ( 2169) occurs as a result of the extremely
negative residuals, and the kurtosis is accordingly high
(1031), meaning that the distribution has a sharp peak
and long thick tails. The residuals for flying squirrels and
New World monkeys make up the most negative tail of
the distribution (their residuals are between 2 and 300
times larger than all species with negative residuals) and
that for meadow voles makes up the most positive tail (35
times larger than the next largest positive residual).
ANCESTOR STATE RECONSTRUCTION
To examine the likelihood that the character state exhibited by flying squirrels and New World monkeys (high
total GC and low MCBC) evolved uniquely and independent of the ancestral species, we performed an ancestor
state reconstruction (Fig. 5) based on the log-transformed
proportion of bound GC (log MCBC log total GC;
referred to hereafter as ‘proportion(s) bound’) (data in
Table S1, Supporting Information). Proportions bound
referred to in the text, tables and figures, however,
represent the untransformed values.
The vast majority of species have proportions bound
that indicate they closely follow convention (yellow, green
and blue branches), and this supports our quantification of
deviations from expected above. This phylogenetic analysis
reinforces the unusual physiology of the flying squirrels
Table 1. Seasonal glucocorticoid (GC) levels and binding capacities for both sexes in adult northern (NFS) and southern (SFS) flying
squirrels
Species
Sex
Season
Dates
NFS
M
Mating/Early
breeding
Mid-late
breeding
Nonbreeding
May/June-2008
M
M
F
F
F
SFS
M
M
M
F
F
F
Mating/Early
breeding
Mid-late
breeding
Nonbreeding
Mating/Early
breeding
Mid-late
breeding
Nonbreeding
Mating/Early
breeding
Mid-late
breeding
Nonbreeding
Total GC
(ng mL 1)
MCBC (ng mL 1)
Free GC
(ng mL 1)
% Free
N
81648 10540
6133 754
77115 10270
9445
13
July/August-2008
119767 10750
12523 1453
107340 10090
8962
8
September
–December-2008
May/June-2008
109948 7485
10471 1453
99559 6478
9055
9
79276 9346
5068 710
74262 8651
9368
6
July–September-2008
82308 9067
5225 270
77142 9187
9372
5
October
–December-2008/2009
May-2008
114682 7752
8149 1108
106694 6774
9295
5
124311 20670
7732 1554
116635 1973
9382
6
June–August-2008
143843 9871
6485 881
137396 9334
9552
7
October/November-2008
May/June-2008
201429 6656
152861 17940
12838 3171
9709 2741
188645 4599
143202 15310
9365
9368
3
8
July–October-2008
168689 24610
8062 2933
160664 2169
9524
4
November-2008/2009
180987 26260
13469 1583
167583 2472
9259
3
M, male, F, female; MCBC, maximum corticosteroid binding capacity.
Data represent means SE. Per cent free GC (% Free) is calculated as (Free GC/Total GC) 9 100.
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
112 L. M. Desantis et al.
(b)
(a)
Fig. 2. Saturation binding curves for (a) flying squirrels and (b) red squirrels. Saturable specific binding was achieved in red squirrel
plasma, but not in flying squirrel plasma due to low affinity of corticosteroid-binding globulin (CBG) for cortisol in this species. Total
binding = cortisol bound to both CBG and serum albumin; Non-specific binding = cortisol bound only to serum albumin; and Specific
binding = cortisol bound only to CBG.
and the New World monkeys (orange branches). It demonstrates that the low-proportion-bound character state
exhibited by these groups evolved from an ancestral state
that followed convention, and that their closest relatives
inherited the ancestral form of the trait; the closest kin of
flying squirrels (all tree and ground squirrels) and of New
World monkeys (all Old World primates, humans and
lemurs) have yellow branches. The brown lemming (Lemmus trimucronatus) and the mud snake (Farancia abacura)
have similarly evolved the low-proportion-bound character
state and done so independent of their ancestral species.
Many fish also possess this character state but, in contrast
to the above, have inherited it from their ancestors. Proportions bound for these species (those represented by
orange branches) are listed in Table 2 along with details
about their closest relatives and the common ancestor.
Proportions bound are extremely low, ranging from 05%
to 24% indicating that these species bind almost none to
very little of their GC. All species differ markedly from
their closest relatives, with the exception of some fish: the
common carp (Cyprinus carpio), who has a proportion
bound similar to Atlantic cod (Gadus morhua); the sea
lamprey (Petromyzon marinus), whose closest relative, the
black-tipped shark (Carcharhinus limbatus), also has a lowproportion bound; and the Pacific hagfish (Eptatretus
stoutii), whose closest relative is the sea lamprey.
Examining all species in the ancestor state reconstruction as a group, the following evolutionary patterns are
evident. The most ancient vertebrates, the hagfish and the
sea lamprey, show very low proportions of bound GC.
More recently evolved fish appear to have inherited this
character state from them, except for a few species (fish
with yellow branches). However, the South American
lungfish (Lepidosiren paradoxa), the closest extant relative
to the tetrapods, has a large proportion bound (342), and
therefore a binding capacity in excess of its hormone
Fig. 3. Scatter plot for 91 vertebrate species
with known plasma maximum corticosteroid binding capacity (MCBC) and total
glucocorticoid levels. The trend line representing a stress axis with 90% binding of
total glucocorticoid was calculated using
the median Kd value of 13 nM
(y = 09x + 3267.Kd). Inset shows a log–
log scale and is used for visual clarity of
individual data points. Abbreviations for
outlier species are as follows: NFS, northern flying squirrel; SFS, southern flying
squirrel; SqM, squirrel monkey; BM,
brown marmoset; WM, white-lipped marmoset; SM, silver marmoset; MV, meadow
vole.
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
Mediating free glucocorticoid in vertebrates
Fig. 4. Frequency distribution of residuals from the 90% bound
trend in Fig. 3. Positive residuals represent points above the trend
line (species with excess binding capacity), and negative residuals
those below it (species with insufficient binding capacity). Species
labels are as those in Fig. 3.
levels. Interestingly, virtually all semi-aquatic and terrestrial species (amphibians, reptiles, birds and mammals)
also show moderate to very high proportions bound and
thus have low levels of free GCs.
Discussion
The majority of vertebrates possess CBG levels and binding affinities typical of those elucidated by biomedical
research, with binding capacities high enough to keep
about 90% of their total GCs out of circulation (Fig. 3
and Table S1, Supporting Information). This implies that
the tissue glucocorticoid receptors (GR) in most species
are highly sensitive to activation by free hormone and that
the metabolic clearance rate of their respective free GCs
may be relatively high. In contrast, North American flying
squirrels and the New World monkeys of South America
have total GC levels higher than all other vertebrates, coupled with extremely low CBG levels (Figs 3 and 4; Table
S1, Supporting Information) and binding affinities (Fig. 2
and Table S1, Supporting Information), resulting in only
10% of their GCs being bound. Both groups evolved this
character state independent of their ancestral species and
thus differ markedly from their closest relatives (Fig. 5;
Table 2). Our study raises two key issues: first, how have
flying squirrels (and the four New World monkey species)
avoided paying the physiological costs of having high free
GC levels and second, how does our ancestor state reconstruction give insight into the origin of CBG? We address
these after discussing caveats that could have affected the
data contributing to the phylogenetic analysis.
The values given in Table S1 (Supporting Information)
and on which Figs 3–5 are based could have been affected
by two factors. First, the stress experienced by the animals
during blood collection could have caused variation in
113
total GC levels relative to their CBG levels. GC levels
increase within 2–5 min of a stressor such as capture and
handling, whereas CBG levels normally decline only in
response to sustained stressors (see references in Delehanty
& Boonstra 2009; Malisch & Breuner 2010). Some of the
values presented in Fig. 3 represent true baseline levels
from unstressed animals (e.g. laboratory and domestic animals, and some bird species caught in mist nets), whereas
others represent levels from animals stressed by capture
and handling. Second, differences between the sexes and
reproductive states could have accounted for some of the
variation in GC and CBG levels. For example, breeding
male mammals often have high testosterone levels and
lower CBG levels relative to non-breeders (e.g. Boonstra &
Singleton 1993). In contrast, in male birds, which lack a
specific sex hormone-binding globulin as found in mammals, high testosterone levels in the breeding season can
increase both GC and CBG levels (e.g. Klukowski et al.
1997; Deviche, Breuner & Orchinik 2001). In female mammals, CBG levels increase during pregnancy as levels of
oestrogen rise (e.g. Seal & Doe 1963; Coe et al. 1986; Selcer et al. 1991). As indicated in the methods, we attempted
to standardize the data we included as much as possible.
Despite the variation these factors could have introduced
to the data, we do not think they markedly affected the
overall pattern of results as the vast majority of species
have residuals within 400 units of the trend line
(Fig. 4).
COPING WITH HIGH FREE GC LEVELS
Both species of flying squirrels and the four species of New
World monkeys have most of their plasma cortisol in the
free state. This presents a fundamental biological dilemma
– how have they avoided paying the costs of having such
high free GC levels when compared with other vertebrates?
We first explore what the normal costs of high free GC
levels are and then how these squirrels and monkeys have
avoided paying them.
Chronically high levels of free GCs are seen in two situations: first, because they are associated with a semelparous
life history, and second, because of a severe, long-term
environmental stressor. Some species have high free GC
levels in the breeding stage of their lifespan (salmonids and
dasyurid marsupials), followed by death. Prior to breeding
free GC levels are low, but at reproduction, these become
chronically elevated. In male dasyurid marsupials, the
consequences are stomach ulcers, anaemia and immune
and inflammatory response suppression (e.g. Bradley,
McDonald & Lee 1980; McDonald et al. 1981). Similar
changes are seen in male salmonids (Idler & Freeman
1968; Barry et al. 2001; Fast et al. 2008) and in partially
semelparous male arctic ground squirrels (Boonstra,
McColl & Karels 2001). Some species or individuals within
these species experience periods of high free GC levels
because of chronic environmental stressors (e.g. high predation risk, social conflict, periods of severe weather),
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
114 L. M. Desantis et al.
Fig. 5. Ancestor state reconstruction of maximum corticosteroid binding capacity (MCBC) as a proportion of total glucocorticoid (logtransformed) for 91 vertebrate species. Orange branches represent species with the lowest proportions bound (i.e. are the most deviant
from the norm). Yellow branches represent species whose proportions bound closely surround a proportion of 09 (or 90% bound; untransformed), including proportions that exceed 10, meaning that they have a binding capacity that can accommodate a large proportion
or all of their total hormone levels. Green and blue branches represent species whose proportions bound are much higher than 10, and
therefore, whose binding capacities greatly exceed their total hormone levels. Branch lengths do not represent evolutionary time.
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
Mediating free glucocorticoid in vertebrates
115
Table 2. Untransformed proportions bound (MCBC Total GC) for species showing orange branches (lowest proportions) in Fig. 4,
compared with those of their closest relative(s). Information about whether the proportion bound of the species of interest differs from the
common ancestor is indicated. Flying squirrels and New World monkeys are referred to collectively as groups
Proportion
bound (log
transformed)
Proportion
bound
(untransformed)
Proportion(s) bound in
closest relative(s)
(untransformed)
Description of
relative(s)
Differ from
common
ancestor?
068
010
094, 106
Tree squirrels
Yes
Species
Common name
Glaucomys
sabrinus
Glaucomys
volans
Callithrix
argentata
Callithrix
jacchus sp.
C. jacchus sp.
Northern flying
squirrel
Southern flying
squirrel
Silver marmoset
057
005
094, 106
Tree squirrels
Yes
041
001
048–116
Old world primates
Yes
Brown marmoset
049
003
048–116
Old world primates
Yes
White-lipped
marmoset
Squirrel monkey
029
001
048–116
Old world primates
Yes
053
004
048–116
Old world primates
Yes
Titi monkey
047
005
048–116
Old world primates
Yes
Capuchin monkey
054
012
048–116
Old world primates
Yes
Brown lemming
069
015
085, 447
Prairie, meadow vole
Yes
Mud snake
Common carp
Atlantic cod
Black-tipped shark
067
047
022
053
024
005
003
021
041, 250
003, 080
080
049
Water, garter snake
Atlantic cod, salmon
Chinook salmon
Southern sting ray
Yes
No
No
No
Sea lamprey
026
001
021, 049
Shark, sting ray
No
Pacific hagfish
067
022
001
Sea lamprey
No
Saimiri
sciureus
Callicebus
moloch
Cebus
albifrons
Lemus
trimucronatus
Farancia abacura
Cyprinus carpio
Gadus morhua
Carcharhinus
limbatus
Petromyzon
marinus
Eptatretus
stoutii
leading to sublethal effects. These effects may include
reduction of fecundity, reduced growth and condition, gluconeogenesis, suppression of immunity and negative effects
on nutrition (e.g. Creel 2001; Cyr & Romero 2007; Sheriff,
Krebs & Boonstra 2009; Zanette et al. 2011). However,
our year-long field trapping program found no evidence of
flying squirrels paying any of these costs (L. Desantis &
R. Boonstra, unpublished data).
The primary explanation for these high free GC levels is
that tissue receptors in the flying squirrels and New World
monkeys do not respond to free GC as they normally do
in other species, and thus, the biological consequences are
not experienced. In the New World monkeys, Scammell
et al. (2001) found that tissue GRs have markedly reduced
affinity for cortisol due to an over expression of the
FKBP51 immunophilin, a protein that forms part of the
GR molecule. This GC resistance means that the high circulating levels of cortisol are necessary to compensate for
the lack of affinity. To bind any hormone at all for tissue
response or for negative feedback to function properly in
the brain, GRs must be completely flooded by free hormone. Scammell et al. (2001) hypothesized that either
increased immunophilin expression in GRs was an adaptation for a system other than the stress axis or that the
mutation may have occurred to counteract the effect of
food rich in GC activity. They suggested that in response,
the stress axis was able to compensate for the incidental
reduction in GR affinity without deleterious consequences
(i.e. by increasing circulating levels of GCs), permitting
these species to persist and radiate within a new habitat.
This explanation is supported by the pronounced dexamethasone resistance in these New World monkeys
(Chrousos et al. 1982). Dexamethasone, an artificial GC,
should cause natural GC levels in the blood to fall rapidly
because of negative feedback at the level of the pituitary.
However in dexamethasone-resistant animals, feedback
fails to function, and GC levels remain high. We also
found flying squirrels to be dexamethasone resistant (L.
Desantis & R. Boonstra, unpublished data), which provides corroborative evidence that similar changes in GRs
may have occurred in these two North American squirrel
species.
An alternative hypothesis to that of an initial change in
the expression of the GR molecule is that a mutation first
occurred at the level of CBG synthesis, rather than at the
level of the receptors; possibly that an environmental factor in the New World caused a genetic defect in the expression of CBG in the liver. The molecular weight of the
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
116 L. M. Desantis et al.
CBG molecule in New World monkeys is about twice that
of humans, Old World primates, rats, rabbits and guinea
pigs (Klosterman, Murai & Siiteri 1986), likely because it
circulates as a dimer (Hammond et al. 1994). It also has a
highly reduced affinity for GCs (Robinson, Hawkey &
Hammond 1985; Klosterman et al. 1986; Rosner et al.
1986). Among other activities, the liver detoxifies and
metabolizes undesirable compounds in the vertebrate body.
Possibly the ingestion of certain phytochemicals or other
toxins initially encountered in the New World, or a novel
parasite infecting the liver or toxin produced by that parasite led to heritable damage or mutation of the DNA
responsible for CBG synthesis. A heritable mutation causing alterations in the structure of CBG has been found in
the BioBreeding strain of laboratory rats well known as
models of autoimmune diabetes mellitus (Smith &
Hammond 1991). The entire breeding strain produces CBG
with an affinity for GCs that is only about half of that in
the more common Wistar rats. Thus, a genetically mutated
CBG molecule that may have persisted over time and radiated within New World monkeys and flying squirrels is
conceivable. The result was the reduced expression and/or
affinity of the CBG molecule, and as a consequence, the
animals lost their GC buffering system in the blood and
adapted by evolving tissue receptors with reduced GC affinity to avoid the deleterious effects of constant exposure to
free GCs. High levels of GC production would still be
needed in a compensatory manner under this scenario, to
achieve any effective binding to receptors when biological
activity was required. Basal GC levels would therefore be
maintained at a high set point to ensure binding to GRs
when the adrenals secrete additional GCs during the stress
response.
If the reduced affinity of both GRs and CBG in New
World monkeys is also correct for flying squirrels, then the
primary functions of CBG (i.e. to act as a buffer and reservoir for GCs) may not hold true in these species. This
leaves us to ask why they would continue to produce the
protein at all. Given that evidence for additional roles of
CBG has been shown in inflammation (e.g. Pemberton
et al. 1988; Petersen et al. 2006) and in extracellular binding of the CBG–GC complex for signal transduction
(reviewed in Hammond 1995), it is likely that these two
groups require CBG for these purposes. Flying squirrels
and New World monkeys would therefore be useful comparative models to further assess alternative roles of CBG.
ANCESTOR STATE RECONSTRUCTION
The phylogenetic analysis indicates that low proportions of
bound GC (MCBC total GC) occur in five groups of
vertebrates: several primitive fish, a reptile (the mud
snake), a cricetid mammal (the brown lemming), four New
World primates and two North American sciurids (the
flying squirrels). Most fish species in our phylogenetic tree
(Fig. 5), starting with the most ancient – hagfish and lamprey, have very low proportions of MCBC. Low specific
GC binding (to steroid-binding proteins) has been found in
other fish species as well (Idler & Freeman 1968; Caldwell,
Kattesh & Strange 1991). Fish do not appear to possess a
true CBG molecule as found in higher vertebrates (and
some marsupial mammals may even possess two CBGs
[Sernia, Bradley & McDonald 1979]), but their GCs may
bind to a protein with lower affinity that simultaneously
binds sex steroids with high affinity (Idler & Freeman
1968; Caldwell, Kattesh & Strange 1991; Breuner &
Orchinik 2002). Baker (2002) hypothesized that the binding
protein in the earliest chordates was albumin. It is found in
the lamprey (Gray & Doolittle 1992), a cyclostome that
arose close to the origins of vertebrates. Baker (2002) suggested that albumin was likely the principle regulator of
steroid access to receptors in primitive fish and played a
large role in protecting steroid receptors from occupancy
by phytochemicals, xenobiotics and chemicals formed by
fungi, bacteria and geochemical reactions at hydrothermal
vents. However, in higher vertebrates, albumin has high
capacity but low affinity for GCs (Peters 1996), and thus,
it does not prevent GC from being functionally free
(Slaunwhite & Sandberg 1959; Westphal 1967).
Corticosteroid-binding globulin has evolutionary origins
separate from albumin, as it is a member of the serine protease inhibitor (SERPIN) family of proteins (Hammond
et al. 1987). SERPINs in general are found widely in metazoans and in the plant kingdom (Silverman et al. 2001),
but CBG has only been found in the vertebrates (Baker
2002). To the best of our knowledge, the exact timing of
CBG’s diversification from other SERPINs remains to be
resolved. Metcalf, George & Brennan (2007) have shown
that the albumin molecule of the lungfish, the closest
extant relative of the tetrapods, is much more similar to
the albumin of tetrapods than it is to that of teleost fish.
This supports our ancestor state reconstruction showing
that lungfish exhibit a character state similar to that of
amphibians, reptiles, birds and mammals, but distinct from
most other fish. Seal & Doe’s (1965) finding in lungfish of
high specific binding that exceeded GC levels by 342
times, whereas all other fish had specific binding that was
only 049 times (or less) GC levels suggest that lungfish
were among the first organisms to evolve a more specific
CBG. This trait was then passed on to the tetrapods and
appears to have been an adaptation for semi-aquatic and
terrestrial life (possibly being related to breathing oxygen
from air instead of water), as most tetrapods (except the
outliers discussed above) show moderate to very high proportions bound. These comparisons should be useful in
furthering our understanding of both functional and evolutionary aspects of stress physiology and the use of steroidbinding proteins in vertebrates.
FUTURE DIRECTIONS
Our research suggests several avenues that warrant investigation. First, how do flying squirrels cope with high levels
of free GC? We need to assess the GC binding affinities of
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119
Mediating free glucocorticoid in vertebrates
brain and tissue receptors at the cellular level. Second, we
need to carry out comparative molecular investigation of
the structure of GC receptors and the CBG molecule in
flying squirrels to determine both how similar their stress
axis adaptations are to those in the New World monkeys
and how their physiology maps on to their behaviour and
natural history. Third, are Old World flying squirrels similar to those in the New World or has divergence occurred
in the latter? If such a divergence is found between the two
groups of flying squirrels, as it has been among the primates, it may indicate that something in the New World
has precipitated this unique adaptation. Fourth, many of
the primitive fish species appear to rely upon albumin for
steroid regulation to a larger extent than do the lungfish
and the higher vertebrates. It is possible that flying squirrels and New World monkeys also rely on albumin for
delivery of GCs to target tissues. Knowledge of albumin
concentrations and affinities for GCs across the vertebrates
would help to clarify the patterns seen here.
Acknowledgements
This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (RB, JTW), the Government of
Ontario (LMD) and the Department of Indian Affairs and Northern Development (BD). We thank G. Keresztesi and J. Middleton for assistance in
the field and laboratory, E. Sager for accommodations and laboratory
space at Trent University’s James McLean Oliver Ecological Centre, J.
Bowman and C. Garroway for advice on trapping flying squirrels and use
of field sites and equipment, and M. Timonin and C. Willis for their
hormone data from little brown bats.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Appendix S1. List of references for published sources used in
Table S1.
Table S1. Glucocorticoid and binding capacity data with related
particulars from published and unpublished sources.
Received 19 June 2012; accepted 31 October 2012
Handling Editor: Charles Fox
© 2013 The Authors. Functional Ecology © 2013 British Ecological Society, Functional Ecology, 27, 107–119