Reviews in Fish Biology and Fisheries 9: 211268, 1999.

1999 Kluwer Academic Publishers. Printed in the Netherlands.

211

Cortisol in teleosts: dynamics, mechanisms of action, and metabolic

regulation
Thomas P. Mommsen1, Mathilakath M. Vijayan2 & Thomas W. Moon3
1 Department

of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada, V8W 3P6;
of Biology, University of Waterloo, Waterloo, Ontario, Canada; 3 Department of Biology, University
of Ottawa, Ottawa, Ontario, Canada (E-mail: tpmom@uvic.ca)
2 Department

Accepted 8 April 1999

Contents
Abstract
Abbreviations
Introduction
Dynamics of cortisol
Biosynthesis
Secretion
Plasm-binding proteins
Metabolic clearance
Uptake and catabolism
Mechanism of action of cortisol
Tissue cortisol receptors
Mammalian corticosteroid receptors
Fish corticosteroid receptors
Receptor compartmentation
Gene regulation
Glucocorticoid receptor regulation
Nongenomic actions
Metabolic regulation
Carbohydrates
Protein and amino acids
Protein turnover
Amino acid metabolism
Ammonia output
Enzymes
Glutamine synthetase
Aminotransferases
Arginase
Other effects
Metyrapone, RU 486 and dexamethasone
Metyrapone
RU 486
Dexamethasone
Lipids
Interactions with other hormones
Physiological role of cortisol
Glucose regulation during stress
Toxicant exposure
Osmoregulation
Growth and reproduction
Future directions
Summary
Acknowledgements
References

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Key words: cortisol biosynthesis, gluconeogenesis, hormone responsive elements, metabolic regulation,
physiology, steroid receptor

Abstract
Cortisol is the principal corticosteriod in teleost fishes and its plasma concentrations rise dramatically during
stress. The relationship between this cortisol increase and its metabolic consequences are subject to extensive
debate. Much of this debate arises from the different responses of the many species used, the diversity of
approaches to manipulate cortisol levels, and the sampling techniques and duration. Given the extreme differences
in experimental approach, it is not surprising that inconsistencies exist within the literature. This review attempts
to delineate common themes on the physiological and metabolic roles of cortisol in teleost fishes and to suggest
new approaches that might overcome some of the inconsistencies on the role of this multifaceted hormone.
We detail the dynamics of cortisol, especially the exogenous and endogenous factors modulating production,
clearance and tissue availability of the hormone. We focus on the mechanisms of action, the biochemical and
physiological impact, and the interaction with other hormones so as to provide a conceptual framework for cortisol
under resting and/or stressed states. Interpretation of interactions between cortisol and other glucoregulatory
hormones is hampered by the absence of adequate hormone quantification, resulting in correlative rather than
causal relationships.
The use of mammalian paradigms to explain the teleost situation is generally inappropriate. The absence of a
unique mineralocorticoid and likely minor importance of glucose in fishes means that cortisol serves both glucocorticoid and mineralocorticoid roles; the unusual structure of the fish glucocorticoid receptor may be a direct
consequence of this duality. Cortisol affects the metabolism of carbohydrates, protein and lipid. Generally cortisol
is hyperglycaemic, primarily as a result of increases in hepatic gluconeogenesis initiated as a result of peripheral
proteolysis. The increased plasma fatty acid levels during hypercortisolaemia may assist to fuel the enhanced
metabolic rates noted for a number of fish species. Cortisol is an essential component of the stress response in
fish, but also plays a significant role in osmoregulation, growth and reproduction. Interactions between cortisol
and toxicants may be the key to the physiology of this hormone, although cortisols many important housekeeping
functions must not be ignored. Combining molecular approaches with isolated cell systems and the whole fish will
lead to an improved understanding of the many faces of this complex hormone in an evolutionary and environmental
framework.
Abbreviations: ACTH adrenocorticotrophic hormone; ANP atrial natriuretic peptide; CBG corticosteriodbinding globulins; CR corticosteroid receptor; CRE cAMP-responsive element; CYP1A1 cytochrome P450
1A1; DBD DNA-binding domain; Dex dexamethasone; ELISA enzyme-linked immunosorbent assay;
EROD 7-ethoxyresorufin-O-deethylase; F1,6BPase fructose 1,6-bisphosphatase; FFA unesterified fatty
acids; FW fresh water; G6PDH glucose 6-phosphate dehydrogenase; GDH glutamate dehydrogenase;
GK glycerokinase; GNSase glutamine synthetase; GPase glycogen phosphorylase; GR glucocorticoid
receptor; GRE glucocorticoid-responsive element; GSase glycogen synthase; HOAD hydroxyacyl-coenzyme
A dehydrogenase; HPI hypothalamic-pituitary-interrenal (axis etc.); HRE hormone-responsive elements; HSD
3-hydroxysteroid dehydrogenase; HSP heat shock protein (hsp 90 etc.); MCR metabolic clearance rate;
MR mineralocorticoid receptor; MSH melanocyte stimulating hormone; -NF -naphthoflavone; NLS
nuclear localization signals; PEPCK phosphoenolpyruvate carboxykinase; POMC pro-opiomelanocortin; PR
progesterone receptor; SERPINS serine proteinase inhibitors and substrates; SW sea water; TA triamcinolone
acetonide; TAT tyrosine aminotransferase; TCBP 3,30 ,4,40-tetrachlorobiphenyl.

213
Introduction
The past decade has seen a flood of information
on the metabolic and physiological effects of stress
in fish and several recent reviews have summarized
these findings (Barton and Iwama, 1991; Gamperl
et al., 1994; Pickering and Pottinger, 1995; Wendelaar Bonga, 1997; Iwama et al., 1998). One of
the most commonly measured indicators of stress
in fish is the concentration of the major circulating
corticosteroid, cortisol (hydrocortisone). The reasons
for this abundance of data on plasma cortisol are
manifold, but, as often, grounded in experimental
accessibility: (1) cortisol can be measured easily
and accurately using commercially available radioimmunoassay (RIA; Gamperl et al., 1994) or enzymelinked immunosorbent assay kits (ELISA; Barry et al.,
1993; Boesgaard et al., 1993); (2) it is possible to
obtain unstressed levels of plasma cortisol by proper
sampling procedure, including anaesthesia (Laidley
and Leatherland, 1988; Iwama et al., 1989); and (3)
plasma cortisol levels tend to increase with exposure
to stressors (see earlier reviews).
However, there are inconsistencies and confusion
in the literature regarding the action of cortisol during stress in fish. Much of the confusion probably
arises owing to differences among species (Vijayan
and Moon, 1994), methods employed to raise cortisol
levels (Gamperl et al., 1994), sampling procedures
(Laidley and Leatherland, 1988; Iwama et al., 1989),
seasonal and diel changes (Bry, 1982; Rance et al.,
1982; Pickering and Pottinger, 1983; Nichols and
Weisbart, 1984; Thorpe et al., 1987), photoperiod
(Audet et al., 1986), nutritional conditions (Vijayan
et al., 1993a; Reddy et al., 1995) and sexual maturity of the fish (Pickering et al., 1987). One of the
underlying assumptions in several studies is that an
elevated plasma concentration of cortisol during stress
is deleterious to the fish, although direct causeeffect
relationships attributed to cortisol have yet to be established (Barton and Iwama, 1991).
Nevertheless, there is increasing evidence suggesting that cortisol directly and/or indirectly plays an
important role in intermediary metabolism (Vijayan
et al., 1994b, 1996a, 1997a), ionic and osmotic
regulation (McCormick, 1995) and immune function
(reviewed by Wendelaar Bonga, 1997), all of which
argues for an adaptive role for cortisol during stress in
fish.
The majority of studies tends to correlate the
physiological changes associated during stress with

cortisol, based primarily on the circulating level of

the steroid. However, plasma concentration of the
steroid is determined to a large extent by the production and plasma clearance of the hormone, i.e. a
sum of dynamic processes, all of which can regulate
the physiological response to cortisol. Indeed, Foster
and Moon (1986) and Vijayan and co-workers (1991)
showed that cortisol-induced changes in tissue metabolism occur even in the absence of increased plasma
cortisol levels.
The objective of this review is to synthesize the
many metabolic and physiological roles of cortisol
reported in fish and to put them into an evolutionary
and environmental framework. In this, we chose to
focus on the teleost fishes, and to address some of the
problems associated with the interpretation of cortisol
effects based on plasma cortisol concentration. We
will restrict our review to the processes involved in
cortisol dynamics and its physiological and metabolic
actions, and will attempt to stay clear of the stress
response in fish, especially because several reviews
on this topic exist already (see above). Nevertheless,
when appropriate, we will discuss the implications of
some of the metabolic and physiological actions of
cortisol in the context of stress in fish.
Owing to the nature of the hormone and the type
of literature available for fish, the current review has a
number of limitations.
1. Information on fish systems is relatively sparse.
2. The literature is generally biased towards the use
of cortisol as an indicator of stress; useful or not,
this has overshadowed its obvious importance in
other physiological processes such as metabolism.
3. The isolated nature of many observations and the
differing experimental protocols employed make
it nearly impossible to derive a coherent picture
for more than the smallest corners of cortisols
domains.
4. Quantification of actual concentration of the hormone (free or physiologically active) is clouded by
the question of the importance and role of cortisolbinding proteins, and whether plasma hormone
concentration is an adequate indicator of hormone effect, compared with estimates of hormone
turnover or clearance.
5. Because correlation does not equal causation, it is
often impossible to identify cortisol as a causative
agent in particular experiments.
6. The absence of specific mineralocorticoids in
fish and their partial functional replacement with
cortisol introduces an interesting dualism for

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cortisol, which applies only to the fishes, with
some startling evolutionary implications.
7. Dexamethasone, an excellent model analogue for
cortisol, is often used to inhibit the hypothalamic
pituitaryinterrenal (HPI) axis, thus indirectly
activating and inhibiting competing routes of
cortisol action.
8. Fish studies are lagging behind mammalian studies
in the availability of antibodies, probes, cDNA libraries and, moreover, owing to the variability and
evolutionary distance of fishes, the usefulness of
mammalian probes is limited.
9. Smoltification is unique to some salmonids and
has no equal in the mammals; yet, its potential
to serve as an interesting model for nonstandard
actions of cortisol and to exemplify the breadth of
its workings still needs to be developed fully.
10. Finally, nongenomic actions of cortisol have been
noticed in fishes, yet researchers have been content merely to describe the associated phenomena
without developing their observations into challenging the dogma for general steroid hormone
action.
Basically by default, therefore, the usefulness of
observations as model systems has been limited and
we would like to make the case that researchers working with fishes finally go beyond the descriptive stage
and approach their quarry more in mechanistic terms.
Intrinsically, the structure of our review therefore follows simple, albeit frustrating lines. Often
we must set the stage using mammalian models and
concepts. We describe similar observations for the
fishes, always happy to point out the uniqueness and
usefulness of fish models, before stating, rather laconically and repetitively, that more work needs to be
done on the fish systems. Yet, what is patently obvious is that fish are highly underutilized models, not
only from an evolutionary point of view, although
at least for developmental biology zebrafish (Danio
rerio, Cyprinidae) have started to fill an obvious void.
Unfortunately, for our specific task, zebrafish are
too small to deliver data on plasma cortisol dynamics, metabolic clearance rates, amino acid utilization,
control of glycogen synthesis or similar physiological parameters. Until appropriate molecular probes
become available, larger fish species must be used as
models.
Cortisol is a multifaceted hormone, not only chemically, but especially physiologically and metabolically. First, it is lipid soluble, yet, because of the
presence of binding proteins in plasma, its physiolo-

gically effective concentration may differ substantially

from what chemical analysis reveals. Second, it may
exert its action by different modes of action: one
of them rapid, nongenomic; the other, the genomic
route, slower and generally longer lasting, and experimentally more accessible. Third, cortisol is always
present in vertebrates, even under unstressed conditions, playing housekeeping roles; yet its actions
tend only to be apparent and appreciated when its
concentration and actions go well beyond housekeeping range. Finally, the hormone does not work in a
vacuum, devoid of other hormones, but rather it interacts at many levels, with many other hormones, setting
up a bewildering array of multilevel, multihormone,
multitarget interactions. As expected, the reductionist approach may lead to interesting insights about the
minutiae, but does little to help develop the big picture. With all these limitations in mind, we will try
to compose a coherent picture of what and why and
how our quarry controls key processes in our finned
friends.
Dynamics of cortisol
Despite the interest in plasma cortisol measurement
as an indicator of stress, few studies have actually
measured the kinetics of cortisol in fish. Plasma
cortisol concentrations reflect the net effect of production and plasma clearance of the hormone. High
or low plasma cortisol concentrations may not be a
good indicator of tissue responses to cortisol stimulation. During chronic stress, plasma cortisol falls
back to the resting levels, even though the fish may
still be responding to the stressor (Vijayan and Leatherland, 1990). The clearance from the plasma is
dependent upon binding proteins, target tissue receptors, tissue uptake and catabolism of cortisol. Thus
the animals physiological response to the hormone
may be modulated by the above-mentioned processes.
Factors affecting any of these parameters will in turn
modify the cortisol response in the animal. Therefore,
a good understanding of the regulatory factors that can
modulate plasma cortisol concentration is paramount
to obtaining some perspective on plasma concentration and the physiological responses associated with
cortisol.
Biosynthesis
Fish do not possess a discrete adrenal gland as in
mammals, and the steroidogenic cells called inter-

215

Figure 1. Biosynthesis of cortisol in teleost fishes. The shaded area

represents the mitochondrial compartment, whereas those reactions
occurring in the nonshaded area occur within the cytosolic compartment. Abbreviations: 3-HSD, 3-hydroxysteroid dehydrogenase;
P450s, various forms of cytochrome P450.

renal cells are distributed in the head-kidney region,

mostly along the posterior cardinal veins and their
branches. These steroidogenic cells lie in close proximity to the chromaffin cells (which secrete catecholamines), raising the possibility of a paracrine
control in the release of these hormones (Reid et
al., 1996). The morphology of the interrenals and
the biosynthesis of corticosteriods by these cells have
been reviewed previously) Idler and Truscott, 1972;
Butler, 1973; Sandor et al., 1984). Briefly, the biosynthesis of cortisol in fish is similar to that in mammals and involves the microsomal enzymatic pathways, including 21-hydroxylation (P450c21), 17hydroxylation (P450c17), and 3-hydroxy steroid
dehydrogenation (3-HSD, Figure 1). In addition, fish
possess the mitochondrial inner membrane monooxygenase enzymes, such as the cholesterol side-chain
cleavage enzyme (cytochrome P450scc, desmolase)
and the 11-hydroxylase that catalyses the 11hydroxylation of deoxycortisol/deoxycorticosterone

(cytochrome P450c11) (Lehoux et al., 1972). Recent

studies have also identified an adrenodoxin-like peptide in the interrenal cells of a teleost the Asian
sea bass (Lates calcarifer, Centropomidae) (SampathKumar et al., 1996); this peptide in mammals functions as a shuttle between adrenodoxin reductase and
cytochrome P450scc/P45011 in the mitochondrial
steroidogenic electron-transport chain. The presence
of adrenodoxin-like peptide immunoreactivity in fish
interrenals hints at the possibility of distinct type I and
type II steroid-hydroxylating enzyme systems (cytochrome P450s), as in mammals. As recently reported
for mammals, control of steroidogenesis may lie outside of the reactions mentioned above, and the key
limiting factor may be the transport of cholesterol to
the outer mitochondrial membrane and its subsequent
translocation across the aqueous intermembrane space
of the mitochondria to the inner membrane (Stocco
and Clark, 1996).
Cortisol is present in eggs and larvae of a number
of species, including Japanese flounder (Papalichthys
olivaceus, Bothidae) (De Jesus et al., 1991), chum
salmon (Oncorhynchus Keta, De Jesus and Hirano,
1992; Hwang et al., 1992), Mozambique tilapia (Oreochromis massambicus, Hwang and Wu, 1993), rainbow trout (Oncorhynchus mykiss, Salmonidae), Asian
sea bass (Sampath-Kumar et al., 1997a) and common carp (Cyprinus carpio, Cyprinidae) (Stouthart et
al., 1988a). Cortisol in unfertilized eggs appears to
be largely of maternal origin (Feist et al., 1990) and
is likely to have been transferred into the growing
oocyte adventitiously via vitellogenin, similar to other
lipophilic hormones such as thyroxine (Specker and
Sullivan, 1995). Endogenous production of cortisol is
first detectable 36 h after fertilization in the common
carp (Stouthart et al., 1998a), but is believed to start
after hatching in flounder (De Jesus et al., 1991), chum
salmon (Hwang et al., 1992), tilapia (Hwang and Wu,
1993), rainbow trout (Barry et al., 1995b) and Asian
sea bass (Sampath-Kumar et al., 1997a). While the
existing database is limited, it hints at species differences in the ontogeny of steroidogenic cell function.
Certainly the regulating mechanisms need to be elucidated as does a functional analysis of the presence
and the production of cortisol in early ontogeny.
Although cortisol is the major circulating adrenocortical steroid in fish, several studies have shown that
stress also increases the concentrations of cortisone
in teleost plasma (Weisbart and McGowan, 1984;
Patio et al., 1987; Pottinger et al., 1992). In vitro,
cortisone can be produced by the head kidney (Idler

216
and Truscott, 1972; Butler, 1973), but the majority of plasma cortisone is due to 11-oxidation of
cortisol in other tissues (Patio et al., 1985). For
instance, incubation of 3 H-cortisol with liver slices
of rainbow trout, perch (Perca fluviatilis, Percidae)
or pike (Esox lucius, Esocidae) confirms that cortisol
is rapidly converted into cortisone (Kime, 1978).
In mammals, the reciprocal conversion of cortisone
to cortisol occurs readily and is catalysed by 11hydroxysteroid dehydrogenase; however, this interconversion has not been clearly established in fish
(Donaldson and Fagerlund, 1972). Because the potential roles of cortisone in fish remain to be identified,
this review will focus on the major corticosteroid,
cortisol. To our knowledge, very few studies have
directly examined the environmental regulation of
cortisol biosynthesis in fish. This may be an important area for future research, especially in light of the
endocrine-disruption action of some environmental
contaminants. Indeed, chemicals inducing cytochrome
P450s may compete for the same substrates in the
steroidogenic pathway, thereby altering the corticosteroid biosynthetic capacity. In birds, the adrenotoxic
environmental pollutant 3-methylsulphonyl-2,2-bis(4-chlorphenyl)-1, 1-dichloroethene (MeSO2 -DDE, a
metabolite of the insecticide DDT) decreased plasma
corticosterone (Jnsson et al., 1994). This decrease
is mediated by the bioactivation of MeSO2 DDE by
adrenal cytochrome P450c11, a key mitochondrial
enzyme involved in cortisol biosynthesis. The outcome is an inhibition of steroid hydroxylase affecting
the final steps of corticosterone biosynthesis (Figure 1)
(Lund and Lund, 1995).
Secretion
The secretion of cortisol is under the control of
the hypothalamuspituitaryinterrenal axis (HPI axis;
reviewed by Donaldson, 1981). Adrenocorticotrophic
hormone (ACTH) released from the anterior pituitary
gland is the main secretagogue for cortisol (Table 1).
The control of ACTH secretion has been reviewed
by Lederis and colleagues (Fryer and Lederis, 1986;
Lederis et al., 1993) and suffice it to say that several factors, including hormones, stress (Sumpter et
al., 1986; Balm et al., 1994) and negative feedback of cortisol at the level of the hypothalamus and
pituitary may modulate ACTH secretion in fish, and
consequently cortisol production.
Most studies on cortisol secretion employed headkidney preparations containing the majority of the

interrenal tissue, either in static or superfusion systems

(Table 1). The basal unstimulated release of cortisol
is higher from interrenal tissue obtained from stressed
fish than from unstressed fish. Stressors, including
high stocking density (Patio et al., 1986; Vijayan and
Leatherland, 1990), confinement (Balm and Pottinger,
1995) and toxicants (Brown, 1993), result in higher
basal release of cortisol from the interrenal tissue.
However, -naphthoflavone (-NF), a potent inducer
of cytochrome P4501A1, failed to elicit any increase
in basal cortisol release in implanted fish (Wilson et
al., 1998), suggesting that the type of contaminant and
the duration of exposure may be important factors in
the cortisol release process. Also, the circulating levels
of ACTH, which are modulated by stress, may play an
important role in the regulation of the HPI axis (Balm
and Pottinger, 1995). It is hypothesized that the higher
basal unstimulated release of cortisol in stressed fish
is due to chronic stimulation of the interrenal cells by
the higher concentration of circulating ACTH (Balm
and Pottinger, 1995) a stimulation that is retained
for some time in the isolated interrenals even in the
absence of ACTH.
Interrenal tissue from stressed fish is less sensitive
to ACTH-stimulation than tissue from unstressed fish.
For example, interrenals from brook charr (Salvelinus
fontinalis, Salmonidae) held at high stocking density
had decreased sensitivity to ACTH-induced cortisol
production (Vijayan and Leatherland, 1990). This
study used a static system, so it is possible that
the build-up of cortisol in the incubation medium in
itself may have affected the release of cortisol. High
levels of cortisol in the medium have been shown
to influence interrenal sensitivity in vitro, via an
ultrashort-loop feedback mechanism (Bradford et al.,
1992). A recent study using a superfusion system,
which overcomes cortisol build-up, confirmed that
prior confinement stress resulted in decreased sensitivity to ACTH in rainbow trout interrenals (Balm and
Pottinger, 1995). Together these results suggest that
either ACTH receptor downregulation and/or desensitization associated with the higher ACTH stimulation
in vivo may be responsible for the decreased sensitivity to ACTH in vitro. Very little is known about
ACTH receptor dynamics in fish interrenal cells and
it is likely that ACTH receptor regulation may be
playing an underappreciated role in cortisol production associated with stress. For instance, organochlorine insecticide or -NF completely abolished the in
vitro ACTH-induced cortisol production in fish interrenal tissue (Ilan and Yaron, 1980a; Wilson et al.,

217
Table 1. Cortisol secretagogues other than adrenocorticotrophin (ACTH) in teleostean fishes
Secretagogue

Comments

Reference

-MSH
Thyroxine
Thyroxine
Angiotensin II

Also acts via ACTH

Sensitizes response to ACTH in presmolts
Desensitizes response to ACTH in postsmolts
Direct and synergistic action with ACTH

Growth hormone
Catecholamines

In vivo, but not in vitro

Lamers et al. (1992)

Young (1986)
Young and Lin (1988)
Decourt and Lahlou (1987),
Arnold-Reed and Balment (1994)
Young (1988)
White and Fletcher (1985),
Schreck et al., (1989)
Takahashi et al. (1985)
Arnold-Reed and Balment (1991)
Arnold-Reed and Balment (1994)
Arnold-Reed and Balment (1994)
Gupta et al. (1985),
Wales and Gaunt (1986)
Barry et al. (1997)
Barry et al. (1997)
Barry et al. (1997)
Young et al. (1996)
Schreck et al. (1989)
Ilan and Yaron (1980a,b),
Patio et al. (1986)
Patio et al. (1986)
Decourt and Lahlou (1986)
Decourt and Lahlou (1986),
Kniehl et al. (1987),
Patio et al. (1988)

N-terminal of POMC
ANP
Urotensin I
Urotensin II
Prostaglandin E1

Direct and potentiating effects with ACTH

17, 20-DHPO
17, 20-DHPO
11-Ketotestosterone
11-Ketotestosterone
Salmonid gonadotrophins
Dibutyryl-30 ,50 -cyclic AMP

No effect (rainbow trout)

Suppression (coho salmon)

Forskolin
Calcium
Increased osmolarity

Slight potentiation of ACTH effects

Physiological role questionable

DHPO, dihydroxy-4-pregnen-3-one; other abbreviations may be found in the list on page 212.
Extracellular calcium is essential for ACTH action (Decourt and Lahlou, 1986).

1998). Furthermore, ACTH-induced cortisol production could be suppressed by treating interrenal tissue
with organochlorines in vitro (Ilan and Yaron, 1980a),
implying that the changes in interrenal responsiveness are mediated directly by the toxicants at the
level of the interrenal cells and not by the circulating
concentrations of other hormones such as cortisol or
ACTH. Interestingly, when cAMP was substituted for
ACTH in the above study, the suppressive effect of
o,p-DDD on cortisol output was prevented (Ilan and
Yaron, 1983), raising the possibility that the effect
may be at the level of receptor desensitization more
so than receptor downregulation per se. As far as we
are aware, studies addressing ACTH receptor dynamics and/or the regulation of the signal-transduction
pathways in fish interrenal cells do not exist. Unfortunately, obstacles in this area include the lack of a
distinct adrenal cortex in fish and the difficulty in isolating interrenal cells for in vitro receptor characteriza-

tion. Any study using a whole head-kidney preparation

will, of course, be influenced by the surrounding cells,
including the chromaffin cells. Because Reid and coworkers (Reid et al., 1996) showed that ACTH induces
catecholamine release in fish and epinephrine stimulates cortisol release in mammals (Mokuda et al.,
1992), the possibility of paracrine influence on cortisol
production seems reasonable.
Although ACTH is the primary secretagogue
for cortisol, several other hormones can indirectly
modify cortisol secretion from the interrenal tissue
(Table 1). Some of these hormones may modulate
the ACTH-induced steroidogenesis. The N-terminal
peptide of salmon pro-opiocortin, for instance, exerts
a weak potentiating effect on ACTH-induced interrenal function in rainbow trout (Takahashi et al.,
1985), while angiotensin II and the urophysial peptides, urotensins I and II, stimulate cortisol secretion in rainbow trout interrenals both in fresh water

218
(FW) and in sea water (SW) (Arnold-Reed and Balment, 1994), with angiotensin II acting synergistically with ACTH (Decourt and Lahlou, 1987). The
possibility that urotensin I enhances the steroidogenic action of ACTH in SW could contribute to
the adaptive changes in osmoregulatory mechanisms
necessary for SW acclimation (see Osmoregulation,
below). Atrial natriuretic factor (ANF) may also
assist in the SW acclimation process by increasing
ACTH-induced corticosteroidogenic capacity in rainbow trout acclimated to SW, but not to FW (ArnoldReed and Balment, 1991). In addition to these peptide
hormones, 11-ketotestosterone (11-KT) suppresses
ACTH-induced cortisol production in rainbow trout
interrenal cells (Young et al., 1996). These results
were further corroborated in vivo in rainbow trout
and brown trout (Salmo trutta, Salmonidae) given
11-KT implants and subjected to confinement stress,
suggesting that gonadal steroids also play a role in
the regulation of the cortisol-release process (Pottinger et al., 1996; Young et al., 1996) and thus will
exert a significant effect on the pituitary interrenal
axis.
Some of the hormones above and several other
hormones have been directly implicated in controlling
the interrenal function of fish (Table 1), including
prostaglandin PGE1 , but not PGF2 , -melanocyte
stimulating hormone (-MSH), and salmon gonadotrophins. These effects clearly imply a complex environmental endocrine interaction in the control of
interrenal function in fish. Very little, however, is
known about the significance of these pituitary and
extra-pituitary hormones on cortisol production. One
hypothesis put forward recently was that these pituitary and extra-pituitary hormones may take on prominence in the absence of ACTH stimulation in order to
maintain plasma cortisol concentration (Wilson et al.,
1998). Indeed, plasma cortisol concentration is maintained in -NF fish despite the abolition of interrenal
sensitivity to ACTH in vitro, suggesting an augmented role for other hormones in the functioning of
the interrenal tissue, or a direct impact of environmental factors. The lack of response or muted cortisol
response to stress in contaminant-exposed fish, however, clearly implies an important role for ACTH
in the stress-induced plasma cortisol levels (Wilson
et al., 1998). Finally, an increase in extracellular
osmotic pressure will result in a sharp, but shortlived, increase in the rate of cortisol excretion from
superfused trout interrenal tissue (Decourt and Lahlou,
1986). Activation of adenylyl cyclase and cAMP are

implicated in the steroidogenic actions described, with

extracellular calcium playing an ancillary role in the
process.
Similar to the synthesis of cortisol, the HPI axis
is functional immediately after hatching in numerous species, including tilapia (Hwang et al., 1992;
Hwang and Wu, 1993), milkfish (Hwang et al., 1992),
Asian sea bass (Sampath-Kumar et al., 1997a), yellow
bream, Japanese flounder (De Jesus et al., 1991) and
rainbow trout (Pottinger and Mosuwe, 1994; Barry
et al., 1995b). A recent study presented evidence for
a functional pituitary-interrenal axis prior to hatching
in carp (Stouthart et al., 1998a), while cultured rainbow trout interrenal cells produced significant levels
of cortisol in response to ACTH at the time of hatching
(Barry et al., 1995a). Carp eggs generated ACTH and
-MSH endogenously 24 h after fertilization. Also,
ACTH and -MSH immunoreactivity was present in
the pituitary of chum salmon two weeks prior to hatching, with the staining intensity increasing gradually
during embryonic life and markedly after hatching
(Naito et al., 1984; Saga et al., 1993). While a cortisol
hypo-responsive period as in mammals was suggested for the rainbow trout (Barry et al., 1995b), no
such reduced HPI activation was evident in developing carp (Stouthart et al., 1998a), leaving the question
open whether different experimental protocols or ontogenetic species differences account for these opposing
observations.
The environmental modulation of interrenal function during development has been poorly studied.
Stouthart and co-workers (Stouthart et al., 1998a)
raised the possibility that rearing temperature for
eggs and larvae may influence the induction of the
cortisol response. Further, it appears that xenobiotics can affect the HPI function during development,
because exposure of carp embryos to PCB 126 results in an increase in whole-body ACTH, -MSH
and cortisol levels (Stouthart et al., 1998b). The significance of elevated cortisol during development is
not clear. Some larval fish (1 day after hatching)
increase body cortisol content when transferred to
20% SW, and cortisol-fed larvae were able to survive better when transferred from FW to SW (Hwang
and Wu, 1993), arguing for a role for cortisol in the
SW, but not the FW, acclimation process. Research
is certainly warranted in a number of areas: first,
to assess the environmental control of the HPI axis;
second, to probe the role of elevated (or decreased)
cortisol concentrations on development and growth;
and third, to analyse the effect of maternal exposure

219
to stress (and resultant high cortisol levels) on larval
development.
Plasma-binding proteins
Mammalian plasma contains steroid-binding globulins, a group of glycoproteins that bind steroids
selectively and with higher affinities than plasma albumin or other plasma proteins. The structure of the
corticosteroid-binding globulin (CBG) is unrelated to
sex-steroid-binding globulin or to any other steroidbinding protein, receptor or enzyme, but is a member of the serine proteinase inhibitors and substrates
(SERPINS) superfamily (Hammond, 1995; Seralini,
1996). The idea that CBG is a protease inhibitor or
substrate fits well with its ability to regulate the plasma
distribution and bioavailability of cortisol at its site of
action. Only free cortisol constitutes the physiologically active form and in mammals 9095% of plasma
cortisol is bound to CBG (Fleshner et al., 1995); thus
the overwhelming fraction of cortisol neither reaches
the target tissues nor binds to target receptors. Because
it harbours the bulk of total plasma cortisol, small
alterations in the concentration in CBG will produce
large changes in the levels of free (biologically active) cortisol. The presence of specific CBG-binding
sites on steroid target cells adds a further local level
of control over cortisol concentrations, with potential interactions between CBG and specific proteinases
(Hammond, 1995). Plasma CBG levels increase when
cortisol levels rise, as in chronic stress (Flesher et
al., 1995) or at a site of inflammation (Hammond,
1995; Garrel, 1996). Dietary manipulations, such as
feeding low-fat diets, will increase plasma CBG along
with free cortisol (Garrel, 1996), providing clear evidence that mammalian CBGs modulate levels of plasma
cortisol.
The situation in fishes appears to differ dramatically. Idler and Truscott (1972) found little evidence for a plasma CBG-like protein in a variety of
fish species. Using equilibrium dialysis (indication
of total binding), the steriod bound was found to
be 4050% of the total steroid, but using gel filtration, less than 5% was bound. On the one hand,
binding of cortisol in fish plasma is one hundredth
of what would be expected in the presence of true
CBGs. On the other hand, it is about tenfold greater
than the binding for human serum albumin, and fish
plasma albumin concentrations fall into the same
range as those of mammals (Maillou and Nimmo,
1993). Studies in the ensuing quarter century have

not changed the view regarding the absence of a

CBG in fish, perhaps because very few studies have
actually addressed this issue in fish. Nichols and Weisbart (1985) reported that total cortisol plasma binding
(equilibrium dialysis) decreased when Atlantic salmon
(Salmo salar, Salmonidae) were transferred from FW
to SW, but this was paralleled by a general decrease in
plasma protein. Later studies from the same laboratory (Chakraborti et al., 1987) failed to find binding
in brook trout plasma by a dilution method. Pottinger (1990) was unable to identify specific plasma
cortisol binding in rainbow trout, although others
showed that mature females had higher plasma binding of cortisol than mature males or immature trout
(Caldwell et al., 1991). This latter study used a
centrifugal ultrafiltration isodialysis method, and represents the only study to identify the presence of a
CBG-like protein in fish blood. One wonders whether
the presence of vitellogenin that binds a number of
lipophilic substances adventitiously may have skewed
these results for the maturing female trout. Additional studies using competition analysis are needed
to resolve this issue, and even though plasma sexsteroid-binding proteins exist in fish (Foucher et al.,
1991), further studies on CBG proteins are useful
if only to show that general plasma proteins suffice for limited binding and transport of cortisol in
fish.
Metabolic clearance
The volume of blood cleared completely and irreversibly of hormone per unit time is known as the metabolic clearance rate (MCR). The MCR for cortisol
indicates the availability of the hormone for tissue
function. The clearance of cortisol from the plasma
represents the net effect of tissue capacity for cortisol
uptake and catabolism. As shown above, turnover
and biological actions in mammals are determined
to a large extent by the dynamics of steroid-binding
proteins in the plasma. In the absence of specific
corticosteroid-binding proteins (CBG) in fishes, other
regulatory sites can be expected. The MCR of cortisol
ranges between 45 mL kg1 h1 (American eel,
Anguilla rostrata, Anguillidae) and 270 mL kg1 h1
(mature sockeye salmon, Oncorhynchus nerka, Salmonidae), values that are considerably lower than the
MCR of 449 mL kg1 h1 measured for cortisone
in the sea raven (Hemitripterus americanus, Cottidae)
(Table 2). Many environmental factors will modify
the clearance of cortisol, including stress, ambient

220
salinity, maturity, and nutritional state. Some of these
effects are collated in Table 2, but it should be kept in
mind that the different methods constant infusion or
bolus injection used for the determination of MCR
may have influenced the actual value of MCR. Nevertheless, statements about changes within concurrent
treatments should hold. Independent of experimental
procedures, it is quite obvious that fish plasma cortisol
concentrations are labile and may be modulated by the
clearance of hormone from the circulation. Because
many of the factors listed in Table 2 alter the plasma
clearance of cortisol, these treatments in turn will
exert significant effects on the plasma concentration
of the hormone and its bioavailability. We believe that
an understanding of the factors modulating cortisol
dynamics is essential to appreciate the physiological
consequences of raised or lowered plasma cortisol
concentrations.
Uptake and catabolism
The clearance of cortisol is essentially maintained by
tissue uptake and catabolism. Because of the lipophilic
nature of cortisol, its entry into cells is thought to
occur by passive diffusion. However, recent studies
have provided evidence of carrier-mediated uptake of
steroid hormone in the rat liver (Allera and Wildt,
1992). In fish, only a few studies have examined
the uptake of cortisol into cells. In isolated rainbow trout hepatocytes, evidence was presented for
a low-affinity carrier protein located in the cellular membrane (Porth-Nibelle and Lahlou, 1981), a
conclusion confirmed in recent studies on the same
species (Vijayan et al., 1997b). The attenuation in
the uptake rate of 3 H-cortisol (40%) by trout hepatocytes in the presence of excess unlabelled cortisol,
over and above the expected decline in specific activity, led us to postulate a distinct uptake mechanism for
cortisol (Vijayan et al., 1997b). These data raise the
possibility of a saturable membrane component that
might play a role in cortisol uptake, similar to the
process observed in rat liver cells (Allera and Wildt,
1992).
Once inside the cell, the steroid is bound to a
receptor and activated or it is metabolized and thus
inactivated. Hormone bound to the receptor will eventually be released from the hormonereceptor complex
and will subsequently also be subject to processing.
The major steroid-metabolizing enzymes in rat liver
microsomes are reductases, oxidoreductases and cytochrome P-450 dependent hydroxylases. The presence

of most of these enzymes has been confirmed for

fish. In rainbow trout, for instance, intra-arterial
injection of 3 H-cortisol resulted in the accumulation of metabolites mostly as water-soluble conjugates of polar derivatives of cortisol (Truscott, 1979).
The steroid moieties of the conjugates, released by
-glucuronidase and aryl sulphatase, were identified
in order of their quantitative importance as tetrahydrocortisone, 20-cortolone, tetrahydrocortisol, 5dihydrocortisone, cortisol and cortisone. The enzymes
involved are 4-ene-5-reductase, 3-hydroxysteroid
dehydrogenase, 20-hydroxysteroid dehydrogenase,
and 11-hydroxysteroid dehydrogenase. In contrast
to the situation in mammalian liver, no evidence was
found for 5-reduction or oxidative cleavage of the
side-chain. In vivo, the major biliary conjugated steroids in rainbow trout 4 h after stress were tetrahydrocortisone, tetrahydrocortisol, cortisone, cortisol and
-cortolone in that order of appearance (Pottinger et
al., 1992).
Liver is the key target organ for cortisol disposal
with the hepato-biliary system as the main route for
cortisol clearance (Idler and Truscott, 1972; Redding
et al., 1984; Vijayan and Leatherland, 1990; Vijayan
and Moon, 1994; Wilson et al., 1998). Renal and
branchial routes play subordinate roles in steroid elimination (Idler and Truscott, 1972; Cravedi et al., 1993).
Very little is known about factors regulating cortisol
uptake and metabolism. As cytochrome P450s are key
players in steroid metabolism and some of these P450
genes are also triggered by xenobiotics, it is likely that
contaminants modulate cortisol metabolism. Vijayan
and co-workers (Vijayan et al., 1997b) explored this
possibility by exposing rainbow trout to the PCB congener, 3,30 ,4,40-tetrachlorobiphenyl (TCBP), a potent
inducer of CYP1A, then examined cortisol uptake
and catabolism in isolated hepatocytes. Fish exposed
to TCBP had significantly higher rates of hepatocyte
3 H-cortisol uptake, concomitant with increased metabolism of cortisol, with tetrahydrocortisone as the
major metabolite (Vijayan et al., 1997b). This study
provided the first evidence that contaminants can significantly alter cortisol clearance. The mechanism(s)
involved in this process is not clear, although one
of the hypotheses is that TCBP influences membrane
fluidity, thereby enhancing cortisol uptake rates. A
recent study using -NF, another potent cytochrome
P450 inducer, showed no effect on cortisol uptake
rate or membrane fluidity. This result suggests that
the effect on cortisol uptake rate may be contaminant
specific, but may also be dependent upon the dura-

221

Table 2. Factors affecting metabolic clearance rates (MCR) for cortisol in teleostean fishes
Species

Sockeye salmon
(Oncorhynchus nerka)

Sea raven
(Hemitripterus americanus)

MCR
(mL h1 kg1 )

54
83

127
449

89102
666

American eel
(Anguilla rostrata)
European eel
(Anguilla anguilla)

Coho salmon
(Oncorhynchus kisutch)

Brook trout (brook charr)

(Salvelinus fontinalis)

Rainbow trout
(Oncorhynchus mykiss)

45

28.4 (SW)
20.6 (FW)

5358 (FW)
84 (SW)

236 (FW)
227 (SW)
105118

176213
260

7696

Changes / comments / references

(Donaldson and Fagerlund, 1968, 1972)

Three fold increase in MCR with maturation in immature males
Three fold increase in MCR with maturation in immature females
(Owen and Idler, 1972)
Cortisol constant infusion
Cortisone constant infusion
(Vijayan and Moon, 1994)
No effect of confinement on cortisol MCR
6-Week food deprivation increases cortisol MCR
6-Week food deprivation decreases plasma cortisol
(Butler, 1973)
SW adapted, single injection
(Leloup-Hatey, 1974, 1976; Henderson et al., 1976)
Higher in SW than FW, plasma cortisol unaltered
MCR higher in hypophysectomized eels, together with decreased plasma cortisol
and decreased cortisol synthesis
MCR doubled after stanniectomy together with increased cortisol synthesis;
plasma cortisol unaltered
(Redding et al., 1984; Patio et al., 1985)
MCR higher in SW than FW fish
Plasma cortisol higher in SW than FW fish
Chronic but not acute stress increases MCR in SW and FW
Seasonal increases in MCR correlate with gill Na+ /K+ -ATPase (smoltification)
No correlation of MCR with plasma cortisol
(Nichols et al., 1985; Vijayan and Leatherland, 1990, 1992)
FW to SW transfer increases cortisol titres
FW to SW transfer increases cortisol synthesis, but not MCR
No effect of high stocking density on cortisol MCR
No effect of RU486 on cortisol MCR
(Brown et al., 1986, 1989)
FW to SW transfer increases MCR, and lowers CBP
Total plasma cortisol unchanged
No difference in MCR between fish held at pH 5.0 and pH 7.7
Increased MCR if exposed to aluminium at low pH
(Wilson et al., 1998)
No effect of -naphthoflavone on MCR
Decreased interrenal sensitivity to ACTH with -naphthoflavone

222
tion of exposure and the metabolic state of the animal
(Wilson et al., 1998). The regulation of cytochrome
P450s and steroid biosynthesis and metabolism will be
a very important area of research, especially with the
emerging use of fish as indicator species for endocrine
disrupters in the aquatic environment.
Mechanism of action of cortisol
Corticosteroid hormones are hydrophobic molecules
that travel bound to plasma proteins and act as transcription factors by binding to specific tissue DNA
sequences. To accomplish this action, corticosteroid
receptors (CRs) must be expressed, or a particular
tissue cannot be said to be a target tissue for that
particular hormone. The dogma regarding corticosteroid hormone action in general, and glucocorticoid
action in particular, is that receptors are localized
in the cytosolic and nuclear fractions only, as membrane permeability does not restrict cellular corticosteroid movements. This dogma has been challenged by
mammalian studies, but work on fish lags well behind.
Tissue cortisol receptors
There have been many recent reviews on corticosteroid receptors (CRs) in mammalian systems (Evans,
1988; Burnstein and Cidlowski, 1989; Fuller, 1991;
Gehring, 1993; Bamberger et al., 1996; Beato et al.,
1996; Guiochon-Mantel et al., 1996; Wehling, 1997)
and it is not the object of this review to exhaustively
present these studies. However, some background is
necessary to provide a framework for the literature
concerned with fishes.
Mammalian corticosteroid receptors
Two cytosolic CRs have been demonstrated in mammalian tissues, termed the glucocorticoid receptor
(GR, type II corticosteroid) and the mineralocorticoid receptor (MR, type I corticosteroid). Classically
a type I receptor has a high affinity for the mineralocorticoid aldosterone, while the type II receptor
has a high affinity for dexamethasone, but low affinity for aldosterone (Knoebl et al., 1996). Both GR
and MR are members of the steroid/thyroid hormone/retinoic acid receptor superfamily, the members
of which are phosphoproteins and ligand-dependent
transcription factors (Burnstein and Cidlowski, 1989,
1992; Fuller, 1991; Schmidt and Meyer, 1994). These
receptors share a high degree of sequence homology in the three linearly arranged domains making

up this class of receptors. Each domain has a specific role based primarily on studies using site-directed
mutagenesis. The central domain contains the DNAbinding domain (DBD) with a sequence predicting
a zinc finger structure with two loops stabilizing the
zinc ion. It is this highly conserved DBD region that
binds to hormone-responsive elements (HRE) called
glucocorticoid-responsive elements (GRE) activating
specific regions of nuclear DNA. The carboxyl terminal contains the ligand-binding site and shows a
high degree of homology between species, but less
between receptor types within the family. The amino
terminal is the least conserved region between species, it is of variable length between receptor types
and even for the same receptor in different species,
it is thought to be the site of receptor regulation and
it contains consensus phosphorylation sequences. The
GRs of mammals are between 85 and 100 kDa in mass,
from 775 to 800 amino acids in length and differ significantly from the MRs of the same species (Fuller,
1991; Gehring 1993).
The composition of the GR in vivo has been more
difficult to establish. It is clear that two molecules of
hsp90 (heat shock protein) are absolutely required for
GR folding and ligand binding (Pratt, 1993; Gehring,
1993; Bohen, 1995). Hsp90 binds at the carboxyl terminal and maintains the GR in an inactivated state;
hsp90 mutants may destabilize the GR aporeceptor
and modify GR signalling (Bohen, 1995). The presence of hsp70 and hsp56 in the aporeceptor is less
well accepted and possibly varies between species,
although recent literature tends to support a role for
both in DNA-binding, transcriptional activities and
receptor trafficking (Diehl and Schmidt, 1993; Czar
et al., 1995). Therefore, the functional GR is composed of one receptor polypeptide, two molecules of
hsp90, and one subunit of about 50 kDa, for a total
molecular mass of about 330 kDa (Bohen, 1995).
There is also evidence that alternate splicing of the
pre-mRNA can result in two human GRs, termed GR
and GR, which differ only in the replacement of
the last 50 amino acids of the C-terminal region with
a unique 15 amino acid sequence preventing GR
from binding glucocorticoid hormones (Bamberger et
al., 1996). The presence of this altered form has
significant mechanistic impact.
The binding of ligands to the GR results in receptor
activation (transformation) and a significant change
to the ligandreceptor complex allowing the movement of the complex to the nucleus and DNA binding.
Although localization of receptor in the nucleus in

223
Table 3. Kinetic characteristics of ligand binding to fish tissue glucocortoicoid receptors
Organ/tissue
and species

Ligand

Kd (nM)

Bmax
(fmol per mg protein)

References

Liver
Rainbow trout
(Oncorhynchus mykiss)

Brook trout
(Salvelinus poutinualis)

3 H-TA

36.0
78.0
Vijayan et al. (1997b)
2.75
61.1
Vijayan et al. (1993a)
3 H-Dex
16.9
87.8
Lee and Struve (1992)
3 H-Dex
16.0
82.3
Lee et al. (1992)
Potency: dexamethasone > TA > cortisol >> corticosterone
3 H-Cortisol
5.1
197
Pottinger (1990)
Potency: dexamethasone = cortisol > RU486 >> corticosterone
3 H-TA

3 H-Cortisol

5.6
167
Chakraborti and Weisbart (1987)
0.6
171
Potency: dexamethasone > TA > cortisol >> corticosterone

Mozambique Tilapia
(Oreochromis mossambicus)

3 H-TA

3 H-Cortisol
3 H-Dex

3 H-Dex
Red tilapia
(Oreochromis mossambicus O. niloticus)

66
9

100
121

Kloas et al. (1998)

11.9

Chen et al. (1997)

3 H-Cortisol
Common carp
31
91
Kloas et al. (1998)
3 H-Dex
(Cyprinus carpio)
36
160
Potency: dexamethasone > TA cortisol > corticosterone > aldosterone; both tilapia and carp

Gill
Rainbow trout

3 H-TA

1.4

271

Coho salmon
(Oncorhynchus kisutch)

3 H-TA

0.5
1.0

60
45

Brook trout

3 H-TA

Sandor et al. (1984)

Shrimpton et al. (1995)
Shrimpton and Randall (1994)

3 H-Cortisol

3.2
224
Chakraborti et al. (1987)
Potency: dexamethasone > TA > 11-deoxycortisol >> cortisol > corticosterone >> cortisone

3 -TA
American eel
2.8
188
Chakraborti et al. (1987)
(Anguilla rostrata)
Potency: TA > dexamethasone > cortisol > 11-deoxycortisol > 21- deoxycortisol

Mozambique tilapia

3 H-Cortisol
3 H-Dex

Common carp

3 H-Cortisol
3 H-Dex

32
7

59
125

Kloas et al. (1998)

31
27

112
122

Kloas et al. (1998)

Brain
3 H-Cortisol
4.5
25.4
Knoebl et al. (1996)
Chinook salmon
3 H-TA
(Oncorhynchus tshawtscha)
0.85
22.4
Potency: TA > dexamethasone > cortisol >> RU486 >> corticosterone >> 17-oestradiol

Rainbow trout hypothalamus

3 H-Dex

12.5
Lee et al. (1992)
Potency: dexamethasone = TA >>> corticosterone
3 H-Dex
Rainbow trout hypothalamus
1.22
296
Allison and Omeljaniuk (1998)
Potency: dexamethasone > cortisol > corticosterone > TA = 11- deoxycortisol >> aldosterone >> 17-oestradiol

3 H-Dex
Red tilapia
9.6
66
Chen et al. (1977)
Potency: dexamethasone > cortisol > 11-deoxycortisol >> 17,20-dihydroxy-4-pregnen-3-one

224
Table 3. Continued
Organ/tissue
and species

Ligand

Kd (nM)

Bmax
(fmol per mg protein)

References

Intestine
Brook trout

3 H-Cortisol

4.5

26.6

Chakraborti et al. (1987)

Intestinal mucosa
American eel

3 H-TA
2.3
900
Dibattista et al. (1983)
Potency: TA > dexamethasone > cortisol > 11-deoxycortisol

Kidney
Common carp

3 H-Cortisol
3 H-Dex

58
70

51
82

Kloas et al. (1998)

Muscle
Brook trout

3 H-Cortisol

2.8

8.3

Chakraborti et al. (1987)

Leukocytes
3 H-Cortisol

2.2
41.4
Maule and Schreck (1991)
0.38
37.8
Potency: TA > cortisol > 17-hydroxyprogesterone > cortisone > aldosterone
3 H-Dex
Common carp, PBL
3.8
490
Weyts et al. (1998)
Potency: TA > dexamethasone >> cortisol > cortisone
Chinook salmon

3 H-TA

Erythrocytes
3 H-Cortisol
4.7
0.33
Pottinger and Brierley (1997)
Rainbow trout
Potency: cortisol = dexamethasone >> 11-ketotestosterone >> 17-oestradiol = cortisone
Not done; Dex, dexamethasone; TA, triamcinolone.
Peripheral blood leukocytes.
Value in binding sites per cell.

some systems has not been demonstrated (Akner et

al., 1995), presumably the ligandGRhsp complex
moves to the nucleus to allow for DNA binding (Bamberger et al., 1996; Guiochon-Mantel et al., 1996). The
hsp complex appears to be essential for this transport
(Pratt, 1993) and specifically hsp56 (Czar et al., 1995)
using a protein-transport system (Akner et al., 1995).
The binding of the activated receptor to GREs will
initiate transcription.
Fish corticosteroid receptors
In contrast to the above, little is known concerning
CR structure in fish. Fish are thought to have only
one CR type, unlike mammals which contain distinct
mineralocorticoid and glucocorticoid (MR and GR)
receptors. This one receptor is termed a GR, consistent
with the lack of a significant amount of a unique mineralocorticoid hormone in fish (Ducouret et al., 1995).

Northern blot analysis and other molecular techniques

indicate that GR mRNAs are expressed in a large number of rainbow trout tissues, including liver, kidney,
gill, intestine, skeletal muscle and brain (Ducouret et
al., 1995; Teitsma et al., 1997, 1998). These methods
supplement earlier studies identifying CRs using competitive binding assays for different ligands, in tissues
such as gills, liver, brain, intestinal mucosa, leukocytes, and erythrocytes of a number of fish species
(Table 3).
The structure of the GR, its association with hsps
or the existence of multiple forms has yet to be
established. The rainbow trout GR (rtGR) is 758
amino acids in length (similar to human GR, hGR)
and has high sequence homology with hGR; this
includes 90% homology within the DBD and 70%
in the glucocorticoid-binding domain. However, the
rtGR contains an expanded region within the zinc

225
finger sequence (Ducouret et al., 1995). This nine
amino acid insert is located between the two zinc
finger sequences, extending this region, while retaining high homologies within the zinc finger sequence
itself (Takeo et al., 1996; Tagawa et al., 1997). The
insert is encoded by a unique exon not found in the
human gene. This modified gene is expressed in all
tissues assayed in the trout, although testes express
both this extended form and a form that lacks the
nine amino acid insert, making this latter form more
similar to other vertebrate GRs (Takeo et al., 1996;
Tujague et al., 1998b). The functional significance of
this insert is not clear. Using cotransfection assays in
CHO cells, Tujague and co-workers (Tujague et al.,
1998b) noticed that the insert does not affect transcriptional efficiency, but it may affect constitutive activity
because cells with the insert had twofold higher levels
of GR expression than those without. Certainly this
difference in sequence may affect other functions of
rtGR and may possibly be involved in the dual functioning of this CR in trout (Ducouret et al., 1995;
Tujague et al., 1998a,b).
There is no report of hsp association with any
fish GR, but given the conserved nature of hsps in
invertebrate and vertebrate species, it is likely that
a GRhsp complex exists in fish species as well.
Evidence in support of the presence of a GRhsp complex is that the cytosol 3 H-cortisol binding activity
of brook trout liver and gill have molecular masses
of 319 kDa (Chakraborti and Weisbart, 1987) and
326 kDa (Chakraborti et al., 1987), respectively, while
American eel gill (Sandor et al., 1984) and intestinal mucosa (Dibattista et al., 1983) are approximately
335 and 230 kDa, respectively. Therefore, their functional molecular masses differ slightly from that of
the complexed heteromeric mass of 330 kDa reported for mammalian GR (Gehring, 1993). Given that
the number of amino acids and the predicted molecular mass of GR in trout (Ducouret et al., 1995) and
human (Gehring, 1993) are similar (85100 kDa), this
additional mass of some 130 to 250 kDa is likely
attributable to hsps or related proteins.
Whether all CRs are GRs or type II receptors in fish
tissues remains to be established. Although Ducouret
and co-workers (Ducouret et al., 1995) reported only
one type of CR in trout tissues with strong homologies to hGR, their specific molecular approach a priori
favoured identification of only GR, not MR. Although
data in Table 3 would support the existence of only a
single GR type, we feel these data are not entirely conclusive. Recepor densities (Bmax ) differ for the various

tissue types, dissociation constants (Kd ) are within the

0.2 to 40 nM range and potency orders always favour
the synthetic steroids dexamethasone or triamcinolone
acetonide. In the few studies employing aldosterone
(Sander et al., 1984; Knoebl et al., 1996; Allison and
Omeljaniuk, 1998; Kloas et al., 1998), binding was
ineffectual compared with the classic glucocorticoids
or their analogues, providing further evidence that the
receptors present in fish tissues group with type II
(GR) and not type I (MR) receptors. Hence, unlike
many mammalian tissues that express MRs and GRs,
fish tissues may have only a single class of receptors.
Additional pharmacological studies using aldosterone
are needed to validate the presence of a single receptor
type in all fish tissues. It should also be noted that
the species listed on Table 3 are primarily salmonids,
and there is evidence for species differences within
mammalian CRs (Fuller, 1991; Burnstein and Cidlowski, 1992; Gehring, 1993). Chen and colleagues
(Chen et al., 1997) using the red tilapia (Oreochromis
mossambicus niloticus) found only a single CR in
all tissues examined. In addition, if as detailed below,
fish skeletal muscle is an important target tissue for
cortisol, the muscle GR needs to be examined, as
the only evidence that such a receptor exists is found
in the molecular studies of Ducouret and co-workers
(Ducouret et al., 1995; Ducouret, 1996).
The brain represents a key site of glucocorticoid and mineralocorticoid function in mammals (Joels
and De Kloet, 1995). With the availability of the
rtGR mRNA, experiments have now been undertaken
to establish expression and distribution of GRs in
the brain of trout. Previous studies demonstrated the
presence of GRs in fish brain (Table 3), but only
the study of Allison and Omeljaniuk (1998) showed
specific binding to a part of the brain (hypothalamus). Teitsma and colleagues (Teitsma et al., 1997)
used rtGR mRNA and Northern blotting and in situ
hybridization to study brain expression and distribution of GRs. Intensive hybridization to the preoptic
nucleus and the nucleus lateralis tuberis, the main
hypophysiotrophic regions in fish, occurred, lending
support to the basic physiological feedback mechanisms reported in many fish species. These studies are
key to a better understanding of the effects and the
interactions of glucocorticoids within fish tissues.
Receptor compartmentation
The subcellular distribution of GRs is controversial
in mammals (Akner et al., 1995; Guiochon-Mantel

226
et al., 1996; Htun et al., 1996) and few studies
have examined this in fish (cf. Knobel et al., 1996).
Evidence supports the nuclear localization of steroid
receptors in the presence or absence of steroid for all
steroid receptors, except the GRs. Translocation of a
receptor protein to the nucleus requires the presence
of nuclear localization signals (NLS) and recognition
of these by binding proteins within the nuclear pore
complex. Studies have identified these NLS to specific
regions between the DBD and the hormone-binding
regions, and if mutated, these receptors are localized to
the cytosol until hormone activated when they move to
the nucleus (Guiochon-Mantel et al., 1996). Although
the mammalian GR has a homologous NLS sequence
aligning with that of the progesterone receptor (PR)
that is localized to the nucleus (Guiochon-Mantel et
al., 1996), Picard and Yamamoto (1987) identified a
region within the carboxyl terminal or ligand-binding
site that was more important. It is believed that upon
GR transformation by ligand the homologous NLS
is critical for translocation. Certainly the binding of
hsp90, which is cytosolic, could protect these translocating sites, potentially restricting the GR to the
cytosol, unlike other steroid receptors. The importance of hsps to the native structure of GR and its
translocation to the nucleus has been reviewed extensively (Gehring, 1993; Pratt, 1993; Akner et al., 1995),
but conflicting views of GR localization continue to
be reported (Brink et al., 1992; Akner et al., 1995)
although the recent visualization of a rat GR bound to
a green fluorescent dye should assist our understanding of this issue (Htun et al., 1996). The prevailing
view is that the exact composition of the GRhsp
complex determines the predominant direction of its
movement (Bamberger et al., 1996).
Working with fresh tissues, Weisbart and colleagues have separated and characterized trout cytosolic and nuclear GRs by using a low-osmotic-strength
homogenization medium and differential centrifugation. Brook trout liver cytosolic GR has a lower dissociation constant (Kd 5.6 vs. 30 nM) and a lower maximum binding capacity (Bmax 167 vs. 858 fmol per mg
protein) than the nuclear GR using 3 H-cortisol as a ligand (Chakraborti and Weisbart, 1987) as does the gill
GR (Kd 3.2 vs 50 nM; Bmax 224 vs 425 fmol per mg
protein; Chakraborti et al., 1987). Similar differences
exist between cytosol and nuclear GRs of rainbow
trout gill (McLeese et al., 1994), and values for fish
GRs fall into comparable ranges to those reported for
rats (Kd 1.2 vs. 65 nM and Bmax 40 vs. 1.1 fmol per mg
protein using 3 H-dexamethasone; Audouin-Chevallier

et al., 1995). The differences between nuclear and

cytoplasmic binding constants and Bmax indicate that
the cytosolic GR has a higher affinity than the nuclear
GR, and in fact Pottinger (1990) has suggested that the
characteristics of the nuclear GRs would be expected
for a nonreceptor-binding protein.
The physiological state of the fish can alter the
compartmental abundance of the GR receptor. In
brook trout gill, for instance, the number of nuclear GRs is increased while that of cytosolic GRs
decreased after SW transfer (Weisbart et al., 1987).
Because plasma cortisol levels were not correlated
to cytosolic GRs, but nuclear cortisol levels were to
nuclear GRs, the authors suggested that the nuclear
GRs were important to SW tolerance in this species.
It is interesting to note that the complex of hsp90
and GR is necessary to display a high-affinity GR
(Simons and Pratt, 1995); as the hsp90 dissociates
from the GR within the nucleus, this low-affinity GR
reported by Weisbart and co-workers may in fact represent a GR devoid of hsp90. Other studies with fish
have failed to quantify nuclear vs. cytosolic GRs, or
have found that nuclear GRs represented a very small
component of the total GR pool, or that the pools
remained unchanged after ligand activation (Lee et al.,
1992; Pottinger et al., 1994; Shrimpton et al., 1995).
This discrepancy between the results of Weisbart and
colleagues and others may represent a simple methodological difference; if not, the fish system may be quite
different from that in mammals and may be an appropriate model system to probe the dilemma of cytosol
vs. nuclear localization for GRs. It should be noted,
however, that most studies on fish report data for total
GR binding and do not distinguish between cytosolic
and nuclear GRs.
Gene regulation
The GR has been considered to be a direct signaltransduction system, in that it binds hormone and
in its transformed state moves to the nucleus where
it affects the transcription of specific genes (Pratt,
1993; Jewell et al., 1995). Although this view is
slowly changing, the principal role of the GR remains
as a transacting transcription factor. The evidence
is that once ligand-bound GR is in the nucleus, it
binds through its DNA-binding domain to one or more
hormone-response elements, termed glucocorticoidresponse elements (GREs), located in the 50 -region of
the promoter of glucocorticoid-sensitive genes. Bamberger et al. (1996) used the term type 1 mechanism

227
to describe this GR action. The GRE lacks specificity
as it can be bound by a variety of steroid receptors
including those for progesterone, androgen and mineralocorticoids (Beato et al., 1996). GR binds as a dimer
and the interaction of the zinc fingers of the DBD
and the GRE with the two trans-activation domains
on GR are probably responsible for the specificity of
gene transcription (Evans, 1988; Fuller, 1991; Beato
et al., 1996). In addition, a series of interactions
with other transcription factors called co-activators
are thought to increase the efficiency of transcription
by RNA polymerase II. The list of co-activators is
increasing rapidly, but already includes TFII and
steroid receptor activator 1 (SRC-1) (Bamberger et
al., 1996) and possibly the cAMP response-elementbinding protein (CREB) (Smith et al., 1996). GREs
and oestrogen-responsive elements are closely related.
For instance, by making one or two base substitutions
(Klock et al., 1987) to this oestrogen-response element, it is possible to confer a glucocorticoid response
to a conserved palindromic sequence in the 50 -flanking
region present in fish, frog and chicken vitellogenin
genes.
A second (type 2) mechanism of GR action inhibits rather than activates transcription (Bamberger et
al., 1996). The promoter lacks a specific GRE, but
GR binds to transcription factors such as Jun and Fos
family proteins and blocks their stimulatory actions on
genes including those of the immune system.
There is no comparable information on any fish
species or tissue. Glucocorticoids have been shown to
modify metabolism and activities of specific enzymes
as well as growth, implying changes in transcription. Given that the GREs are promiscuous, that the
trout GR protein is altered at the zinc finger sequence
(Ducouret et al., 1995), and that fish are devoid of
an unique mineralocorticoid, the fish GR-transcription
system could be important to assist our understanding of the complex evolution of this steroid-receptor
family. This area could also be important in terms of
the possible relationship between the responses of fish
to toxicants such as dioxins, and the stress response
initiated by glucocorticoids. Recently, Celander and
co-workers (Celander et al., 1996) reported that dexamethasone potentiates the actions of aryl hydrocarbon receptor inducers on both cytochrome P4501A
(CYP1A) activities and protein, but not on CYP3A.
This result suggests a specific interaction between
inducer elements and a GRE on the CYP1A gene, and
not only a widespread location of GREs in the fish
genome, but also that the fish system is not unlike that

seen for mammals. For instance, a GRE-like motif

was localized on the rainbow trout prolactin gene
(tPRL) promoter (Argenton et al., 1996). Activation of
tPRL apparently requires the simultaneous occupancy
of both the GRE site and a second site for an additional transcription factor (GHF1). During vertebrate
evolution, the conservation of GHF1 appears critical
for PRL, but what seems to differ are the modulators
of this particular site. Studies in this area may discover some very fascinating evolutionary differences
and may explain the absence of a mineralocorticoid in
teleosts.
Glucocorticoid receptor regulation
The regulation of GR activity has been reviewed for
the mammals (Gehring, 1993; Schmidt and Meyer,
1994; Bamberger et al., 1996). Clearly, the sensitivity of a particular tissue to corticosteroids will be
established by the type and level of receptor expressed
and will also be affected by either down-regulation
or up-regulation of expression. Arriza and co-workers
(Arriza et al., 1988) have also suggested, for mammalian tissues that coexpress GR and MR, that the MR
may function as a high-affinity GR. GR, in turn, may
only be activated at much higher circulating concentrations of corticosteroids. This selectivity mechanism
is in addition to the well-known enzymatic conversion
of cortisol to cortisone by 11-hydroxycorticosteroid
dehydrogenase, permitting aldosterone binding to MR
rather than cortisol (Fuller, 1991). Autoregulation of
GRs is dependent upon the tissue or cell type, developmental stage and the prior corticosteroid history of
the animal/tissue. Homologous down-regulation has
been shown to occur by decreasing the rate of GR gene
transcription (thus reducing GR mRNA and protein),
destabilization of GR mRNA (post-transcriptional)
and reduction in the half-life of GR protein (posttranslational, including phosphorylation). In each
case, it is clear that either the glucocorticoid or the GR
interacts with specific sequences on the GR mRNA or
cDNA. The mechanism of homologous up-regulation
is not understood, but is known in both normal and
adrenalectomized mammals. Heterologous regulation
by other steroid hormones, and in particular the sex
steroids, by regulating levels of GR mRNA, has also
been reported. The cloning of the human GR promoter (Nobukuni et al., 1995) should assist in our
understanding of these regulatory features; certainly
the existence of a large number of sites for transcription factors means that many components could alter

228
GR mRNA levels. Recent studies also indicate the
existence of endogenous, low-molecular-weight modulators (a phosphoglyceride) of corticosteroid receptors (GR and MR) in mammalian liver cytosol (Bodine
and Litwack, 1995), but how these modulators are
related to GR regulation is at the moment unclear.
It is clear, however, that many other components of
the glucocorticoid signal cascade system are modulated by a host of factors which in themselves are
modulated (Bamberger et al., 1996). This complexity
will increase as additional components of the system
become better defined. GR is a phosphoprotein, and
seven phosphorylation sites have been found in the
mouse receptor, all but one in the amino terminal
domain (Kuiper and Brinkmann, 1994). Although the
role of phosphorylation is clear for some of the sex
steroid receptors (e.g., oestrogen), no such evidence
is available for GR. In fact, some feel (Bamberger et
al., 1996) that phosphorylation has no impact on GR
activity, but may be more important to establish cellular localization. This is an obvious area for further
studies.
Again, studies with fish systems have shown GR
autoregulation, but the precise mechanism(s) is commonly not understood. Generally, an inverse correlation is noted between plasma cortisol level and the
number of GRs in fish tissues. This has been clearly
demonstrated in liver by both confinement stress,
which increases plasma cortisol, and injection or
implants containing either cortisol or dexamethasone
(Pottinger, 1990). No such correlation was noted
in trout injected with 3,30 ,4,40-tetrachlorobiphenyl
(TCBP), even though plasma cortisol values increased
by 3.5 fold (Vijayan et al., 1997b). Other studies
found a significant linear correlation in trout between
approximately 1 and 30 ng mL1 cortisol and Bmax
values in control and cortisol-dosed fish, without alterations in the Kd (Pottinger, 1990; Lee et al., 1992).
Similarly, trout brain GR numbers were inversely
related to plasma cortisol (Lee et al., 1992), with
a unique increase in Kd . Carp fed a single cortisolcontaining meal had increased plasma concentrations
of the hormone for one day (Weyts et al., 1998).
This increase was associated with a 50% decrease in
cortisol binding to peripheral blood leukocytes by 3 h
that persisted for more than 4 days even though plasma
cortisol had returned to baseline. The observation that
addition of linoleate (C18:2) to trout liver cytosol
increased the Kd , but did not affect the Bmax for 3 Hdexamethasone binding, was interpreted as indicative
of changes in GR conformation, but the precise mech-

anism is unknown (Lee and Struve, 1992; Lee et al.,

1992). Unsaturated fatty acids inhibit hepatic cytosol
3 H-dexamethasone binding in a mammal by a mixed,
noncompetitive manner (Sumida, 1995), without altering GR mRNA, suggesting that the fatty acids may
be acting indirectly through a protein kinase system.
Because fish fatty acid composition differs from that
of endothermic mammals and can be altered experimentally by thermal adaptation (Hazel, 1993) and by
dietary manipulation (Labbe et al., 1995), fatty acid
control over fish GRs could be an intriguing area for
further research.
The situation with gill GR binding is complicated by developmental stage, especially smoltification in salmonids. As noted elsewhere in this
review, smoltification is associated with increased
plasma cortisol levels, increased gill Na+ /K+ -ATPase
activities, chloride cell proliferation and enhanced
SW tolerance. Generally, numbers of gill GRs are
positively related to the ability of a salmonid for
hypo-osmoregulation, i.e. to undergo smoltification
(Shrimpton et al., 1994). However, a number of studies have reported that both acute and chronic elevation
of cortisol decreased the maximum binding capacity
of gill GRs in salmon and trout (Maule and Schreck,
1991; Shrimpton and Randall, 1994; Shrimpton et al.,
1994; McLeese et al., 1994). It is this decreased sensitivity to circulating cortisol that is thought to interfere
with smoltification in stressed salmonids (Shrimpton
and Randall, 1994). In a related experiment, injection
of the somatotrophic (mammalian) placental lactogen
or (piscine) growth hormone increased both the number of gill GRs and gill Na+ /K+ -ATPase activities
without consistent impact upon plasma cortisol levels
in coho salmon (Shrimpton et al., 1995). These results
were interpreted as indicating that the simultaneous
release of somatotrophins and cortisol during smolitification may ameliorate the direct effects of cortisol
on decreasing gill GRs. Recently Tagawa et al. (1997)
also found increased intensity of GR mRNA in gills
from tilapia raised in FW compared with fish raised in
full-strength SW.
In no case has there been a mechanistic explanation
for down-regulation of any fish tissue GR, although
as mRNA probes and antibodies become available for
more GRs, mechanistic answers will not be far behind.
Sea water transfer in brook trout was associated with
increased numbers of gill nuclear GRs (Weisbart et
al., 1987), suggesting this translocation was critical,
but no other study has confirmed this observation. The
studies using somatotrophins are intriguing (Shrimp-

229
ton et al., 1995), but the authors indicate that the
effects of growth hormone are additive; as a result, upregulation, in this case heterologous autoregulation,
is not an issue. The fact that cortisol has permissive
effects on fish metabolism, as in mammals, may
implicate autoregulation of fish tissue GRs by other
hormones. Studies examining the GR promoter in the
fish system need to be undertaken to achieve a better understanding of which factors may regulate fish
GRs. These molecular technologies have already been
used successfully for other fish promoters, for example
prolactin in the context of glucocorticoid activation
(Argenton et al., 1996) and insulin (Argenton et al.,
1997), and should be readily transferable to the study
of the GR.
Fasting in rainbow trout resulted in increased
amounts of cortisol specifically bound to erythrocytes
(Pottinger and Brierley, 1997). Few similar studies
have been undertaken, although for tilapia, red blood
cells have been shown to express higher levels of GR
mRNA than any other tissue examined (Tagawa et al.,
1997). This study points to a lack of information on
how stressors affect the density of GRs in fish species
and should provide new avenues for future studies on
the control of fish GRs. Now that antibodies have been
produced for the rtGR (Tujague et al., 1998a), we are
closer to more appropriate models for assessing GR
expression under a variety of conditions.
Nongenomic actions
Increasing evidence suggests that steroid hormones,
including glucocorticoids, exert nongenomic effects.
For the mammals, this area has been reviewed
(Wehling, 1997) and the list of steroid hormones and
tissues involved is extensive. Nongenomic effects of
steroid hormones are rapid (seconds to a few minutes).
For instance, the effects of dexamethasone on glycogen metabolism peak within 515 min of exposure
to this analogue (Baqu et al., 1996). The effects are
also insensitive to inhibitors of transcription or protein
synthesis, actinomycin D and cycloheximide, and generally involve membrane actions and changes in intracellular calcium ([Ca2+ ]i ). The best-studied cases are
aldosterone and progesterone actions in mammalian
kidneys and sperm/oocytes, respectively.
The case for nongenomic actions of glucocorticoids is less well established, but, again, supporting evidence is accumulating. Radioligand (3 Hdexamethasone) and immunofluorescence studies led
Grote and colleagues (Grote et al., 1993) to sug-

gest GR distribution to both cytosol and membrane

compartments of rat liver cells. Others were able
to distinguish between cytosol and membrane 3 Hdexamethasone binding by rate of association, differential competition with glucocorticoid agonists, and
sensitivity to the thiol reactive agent arsenite, again in
rat liver (Wright and Paine, 1995). Other studies have
been summarized by Wehling (1997).
Comparable phenomena of rapid action have been
described for cortisol and dexamethasone in fish. In
a tilapia pituitary perifusion preparation, for instance,
the stimulation of prolactin release by a hyposmotic
medium was blocked by cortisol (200 nM) and became
significant compared with controls by 20 min (Borski et al., 1991). In addition, the increase in cAMP
and Ca2+ in these cells to hyposmotic medium could
also be blocked by cortisol within 15 min, implying that cortisol can have a rapid effect and that
this effect is mediated through pathways that modify
cAMP and Ca2+ levels. Similarly, dexamethasone
exerted a short-term tropic effect on a goldfish melanocytoma cell line. Within 20 min of application of the
analogue, cells flatten out and undergo extension and
broadening of dendrites. The effect was specific for
glucocorticoid, as it was blocked by the corticosteroid antagonist RU486. More importantly, however,
the reaction to dexamethasone remained unaffected by
cycloheximide or actinomycin D (Shih et al., 1990),
clearly indicating that the effect was nongenomic and
independent of transcription and protein synthesis. As
in the case of prolactin release (Borski et al., 1991),
Ca2+ seems to play an important role in the rapid
changes in melanocytoma cell morphology (Shih and
Lo, 1993). The observation that 3 H-cortisol binds specifically, albeit to a limited extent at around 8% of
total binding, to trout erythrocyte membranes (Pottinger and Brierley, 1997) provides another indication
that nonstandard mechanisms for corticosteroid action
do occur in fish. It remains to be seen how widespread
these phenomena are and whether they have metabolic consequences. Metabolic studies using cortisol
in fish generally preclude the identification of such
short-term, possibly nongenomic, actions of this hormone. Even so-called acute in vitro hepatocyte studies (Renaud and Moon, 1980; Foster and Moon, 1986)
sample after 1 to 2 h, i.e. well past the time of changes
noted in the tilapia pituitary or goldfish melanocytoma cells, and those particular hepatocyte studies did
not use inhibitors of protein synthesis or transcription. Therefore, careful short-term studies need to be
undertaken, possibly using the hepatocyte system to

230
include the possibility of identifying surface receptors
for cortisol or its agonists in fish cells.

cortisol concentrations, it is clear that carbohydrates,

protein turnover, amino acid dynamics and lipids are
consistently identified as important control points for
this hormone.

Metabolic regulation
Carbohydrates
The treatment of fish with cortisol is even more varied
than the number of species examined. Apart from the
expected in vivo versus in vitro approaches, treatments
have been done with suspected agonists (cortisol,
dexamethasone, triamcinolone), and different modes
of application (single or repeated injection, at differing
sites, oral application, various oil deposits or osmotic
minipumps). These varied approaches are exacerbated
by a bewildering array of treatment times, ranging
from minutes to weeks. Other routes of inquiry have
employed crowding, exhaustive exercise or similar
stress situations with the aim of increasing endogenously produced cortisol concentrations. While this is
not the place to discuss the relative merits of these
different approaches, it must be kept in mind that the
experimental design is likely to strongly influence the
outcome of a particular experiment (Gamperl et al.,
1994). As outlined in the following, this consideration
is nowhere more obvious than in a discussion of glucocorticoid action on carbohydrate, protein and amino
acid metabolism. Furthermore, biological variation
between species will have a bearing on experimental
outcomes, rendering the derivation of common themes
for cortisol action a difficult task, especially for protein
metabolism, which is a priori not easily accessible
experimentally.
A clear differentiation must be made between
short-term, acute effects and long-term treatments
with cortisol. Under acute stress situations, plasma
cortisol concentrations tend to shoot up within a
minute to hour time frame, followed by a gradual
decrease to pretreatment levels within a day or so,
depending upon subsequent maintenance conditions.
Hence, under longer-term stressful situations, the fish
seem to adapt to the stress and plasma cortisol fluctuates within the range considered normal for a particular species. An acute stress can be simulated experimentally with a single injection of cortisol (generally
a stress itself, thus releasing endogenous cortisol; see
below), although the time course of an augmented
cortisol titre tends to be longer than under acute stress
situations. In contrast, long-term exposure to exogenous cortisol with oil or similar deposits usually results
in chronically elevated plasma cortisol concentrations.
Independent of macro- or micro-control over plasma

Glucocorticoids modulate hepatic glucose metabolism in vivo in mammals (Goldstein et al., 1992,
1993) and in isolated hepatocytes in vitro (Jones et
al., 1993), generally stimulating gluconeogenesis and
increasing liver glycogen content. Two key mechanisms have been identified by which glucocorticoids
stimulate gluconegoenesis. The first is induction of
phosphoenolpyruvate carboxykinase (PEPCK) activity by increased transcription (Granner et al., 1986)
of PEPCK mRNA by sixfold, especially in the presence of glucagon, together with a fourfold increase
in the stability of its mRNA. This stabilization is
a process dependent on a glucocorticoid-responsive
element (GRE) in the 30 -noncoding region of the PEPCKmRNA (Heinrichs et al., 1994). At the same time,
glucocorticoids destabilize the mRNAs of other hepatic proteins, including those of interleukin 1 and
3-hydroxymethylglutaryl-CoA reductase. The second
mechanism is a stimulatory effect on hepatic precursor
supply, thereby sustaining gluconeogenesis in vivo
(Fujiwara et al., 1996). Stimulation of glycogen synthesis is thought to be due to the hormone-dependent
activation of glycogen synthase by increasing glycogen synthase phosphatase activity (Vanstapel et al.,
1982), although glucocorticoid stimulation of glycogen synthesis in mammalian liver is not supported
by all studies. At various times, the absence of glycogen synthesis (Hems and Whitton, 1980), reduced
glycogen content and activated glycogenolysis (Baqu
et al., 1996) have been reported. In isolated hepatocytes, glucocorticoid did not stimulate glycogen
synthase, even though glycogen synthase phosphatase
was activated (Laloux et al., 1983). In recent studies
with rat hepatocyte primary culture, dexamethasone
increased both glycogen synthase and glycogen phosphorylase activities without changing their mRNA
content, suggesting that the model glucocorticoid controls the activity of these enzymes by modifying their
phosphorylation state rather than by affecting gene
expression (Baqu et al., 1996). Also, the rapid activation of glycogen phosphorylase with dexamethasone,
peaking between 5 and 15 min after dexamethasone
administration, clearly supports possible nongenomic
effects of glucocorticoid on glycogen metabolism.

231
Table 4. Changes in blood glucose and liver glycogen with exposure to corticosteroids in teleost fish
Change

Species

References

Plasma glycaemia
Increase

Decrease
Unaltered

American eel (Anguilla rostrata)

Japanese eel (Anguilla japonica)
European eel (Anguilla anguilla)
Roach (Rutilus rutilus, Cyprinidae)
Gulf toadfish (Opsanus beta, Batrachoididae)
Sea raven (Hemitripterus americanus)
Mozambique tilapia (Oreochromis mossambicus)
Killifish (Fundulus heteroclitus, Cyprinodontidae)
Coho salmon (Oncorhynchus kisutch)
Cutthroat trout (Oncorhynchus clarki)
Rainbow trout (Oncorhynchus mykiss)
American eel
Brook charr (Salvelinus fontinalis)
Channel catfish (Ictalurus punctatus, Ictaluridae)
Brook charr
Rainbow trout

Butler (1968)
Inui and Yokote (1975), Chan and Woo (1978)
Lidman et al. (1979)
Mller and Hanke (1974)
Mommsen et al. (1992)
Vijayan et al. (1997a)
Vijayan et al. (1997a)
Leach and Taylor (1982)
Vijayan and Leatherland (1989)
Morgan and Iwana (1996)
de la Higuera and Cardenas (1986), Barton et al. (1987)
Foster and Moon (1986)
Vijayan et al. (1991)
Davis et al. (1985)
Tam et al. (1988)
Leatherland (1987), Andersen et al. (1991), Vijayan et al. (1994a)

Liver glycogen
Increase

Decrease

Unaltered

American eel
Japanese eel
European eel
Killifish
Mozambique tilapa
Brook charr
Rainbow trout
American eel
Brook charr
Coho salmon
Rainbow trout
Goldfish (Carassius auratus, Cyprinidae)
Gulf toadfish
Sea raven
Mozambique tilapia
Rainbow trout

The rapid activation of glycogen phosphorylase was

independent of cAMP and completely abolished in
the absence of Ca2+ (Gomez-Munoz et al., 1989).
Despite glycogen synthase activation, the exposed
cultured rat hepatocyte contained less glycogen than
nave controls, indicating that dexamethasone exerts a
net glycogenolytic effect in hepatocytes (Baqu et al.,
1996).
In fish, the majority of studies relating cortisol
effect on carbohydrate metabolism rely on plasma
glucose and liver glycogen content as indicators of

Butler (1968)
Inui and Yokote (1975), Chan and Woo (1978)
Lidman et al. (1979)
Leach and Taylor (1982)
Swallow and Fleming (1970)
Whiting and Wiggs (1977)
de la Higuera and Cardenas (1986)
Foster and Moon (1986)
Whiting and Wiggs (1977)
Vijayan and Leatherland (1989)
Barton et al. (1987), Andersen et al. (1991)
Storer (1967)
Mommsen et al. (1992)
Vijayan et al. (1996a)
Vijayan et al. (1997a)
Leatherland (1987), Pagnotta et al. (1994), Vijayan et al. (1994a)

metabolism. Both liver glycogen and plasma glucose concentrations vary substantially depending on
species, developmental stage and metabolic state of
the animal and, perhaps consequently, the results
are not unequivocal (Table 4). Following exposure
to increased cortisol levels, plasma glycaemia can
be increased, decreased or remain unaltered. A similarly variable response occurred for liver glycogen
(Table 4). Considering these results, it is not surprising
that some confusion has arisen concerning the role of
cortisol on carbohydrate metabolism.

232
Apart from this seemingly random picture of the
role of cortisol, we would like to point out that reliance
on plasma glucose levels and liver glycogen content
may not be a beneficial approach in the first instance.
Fish plasma glucose is highly variable with physiological state, is not as tightly defended against exogenous disturbances as in mammals, and especially for
the many carnivorous species analysed, it may not be
a useful indicator of metabolic status. Similar considerations apply to hepatic glycogen. Although this
parameter is not as volatile as in mammalian liver,
interspecies variability is huge in fishes, as is withinspecies variability, even within closely related groups
of experimental specimens. This latter fact results in
unusually large standard deviations for experimental
groups, unless clonal groups of fish are used (Plisetskaya, 1975; Plaut and Gordon, 1994). Hence, obtaining reliable data on plasma glucose and liver glycogen
as indices of carbohydrate metabolism is a tall order.
In the context of cortisol, the prior rearing history of
the fish, including their nutritional state, may modulate
the response of liver glycogen to cortisol treatment
(Leach and Taylor, 1982; Vijayan et al., 1993a).
Finally, plasma glucose concentration measurements
provide only a static snapshot of the metabolic situation and do not take into account the turnover of
the metabolite, which could be influenced by several
factors and may mask any cortisol effect. Thus, relying on plasma glucose concentration or liver glycogen
content alone will not give a clear picture, unless data
are supported by other measurements, such as enzyme
activities or turnover rates to denote specific pathways
activated by cortisol.
As mentioned above, dexamethasone increases the
percentage of glycogen phosphorylase in the active
form, and thus the overall activity of the enzyme. This
effect in mammalian liver, as well as the concomitant
increases in glycogen synthase and its translocation
into a pelletable form, are directly brought about
by the glucocorticoid and are independent of nuclear hormone actions and protein synthesis (Baqu et
al., 1996). This metabolic setup, basically mimicking
the fasted state, is somewhat reminiscent of the situation in fishes, where both increases in the rate of
glycogen breakdown, hyperglycaemia and increases
in liver glycogen have been reported; however, in a
direct test, we failed to detect activation of glycogen phosphorylase by cortisol in freshly isolated or
cultured tilapia hepatocytes (M. M. Vijayan and T.P.
Mommsen, unpublished data). A similarly confusing result is obtained for rat hepatocytes where, in

the presence of dexamethasone, flux through gluconeogenesis is increased at the same time as pyruvate
kinase (Jones et al., 1993) a scenario composed of
two mutually exclusive processes, potentially resulting
in an increased rate of futile cycling.
Nevertheless, the consensus for fish liver is that
cortisol treatment significantly enhances the rate of
gluconeogenesis, despite the counterintuitive absence
of changes in plasma glucose or even lowering of
plasma glucose (Table 4). This conclusion is based
on the observation that cortisol treatment significantly increases the activities of all key gluconeogenic
enzymes, namely glucose 6-phosphatase, fructose
1,6-bisphosphatase and PEPCK. These and other
enzymes positively controlled by corticosteroids are
collated in Table 7, below. Increased activities of
these gluconeogenic enzymes support an increased
liver capacity for gluconeogensis in cortisol-treated
fish, while the absence of an increase in plasma glucose concentration may result from elevated turnover
of the metabolite. Additional support comes from
experiments on isolated liver systems. Significantly
enhanced rates of gluconeogenesis from lactate were
noted in isolated eel hepatocytes (Renaud and Moon,
1980) and carp liver slices (Janssens and Waterman,
1988) incubated with cortisol in vitro, and in hepatocytes isolated from Gulf toadfish (Opsanus beta, Batrachoididae) previously injected with dexamethasone
(Mommsen et al., 1992). In the case of the toadfish,
this increased responsiveness was not accompanied
by changes in hepatic PEPCK activity, locating the
site of glucocorticoid action in this species away from
this key step in gluconeogenesis. Finally, in cortisolimplanted sea ravens, hepatocytes have increased rates
of alanine oxidation (Vijayan et al., 1993b), at least
alluding to an increased gluconeogenic potential.
Two studies fail to support the above generalization. First, American eels given repeated cortisol
injection in vivo did not show any cortisol-mediated
effect on glucoenogenesis from lactate or alanine in
isolated hepatocytes (Foster and Moon, 1986), but
the repeated handling may have altered gluconeogenic
flux in the control fish, thus masking any cortisolspecific responses. Second, in cortisol-implanted rainbow trout, hepatic PEPCK activity and plasma glucose
turnover rate remained unaltered (Andersen et al.,
1991). These negative data were internally consistent, because the study failed to detect any significant effect of cortisol on direct gluconeogenesis or
oxidation from 14 C-labelled alanine or lactate in isolated hepatocytes (Andersen et al., 1991). Another

233
study in the same species, showed cortisol-induced
hyperglycaemia and increases in liver glycogen, while
gluconeogenesis from glutamate, measured as flux
into plasma glucose, liver and muscle glycogen, was
unaffected (de la Higuera and Cardenas, 1986).
However, more recently, and using a slightly different approach, Vijayan et al. (1994b) showed that
cortisol implantation significantly increased gluconeogenesis and oxidation from 14 C-alanine in rainbow
trout hepatocytes. This stimulatory effect was abolished in the presence of RU486, a glucocorticoid
receptor antagonist, indicating a classical receptormediated effect of cortisol on gluconeogenesis. Considering the multitude of species, their different
responsiveness to cortisol (Vijayan et al., 1993a,
1994b), different methods of cortisol exposure (in
vivo vs. in vitro), different techniques used to elevate cortisol levels, duration of exposure (Gamperl et
al., 1994), nutritional status (Vijayan et al., 1993a),
reproductive state/gender of the animal (Pottinger et
al., 1995, 1996), seasonal effects and prior rearing (stress) history of the fish, it is not overly surprising that some inconsistencies in cortisol action
have arisen. Primary, long-term cultures of hepatocytes avoid much of this exogenous variability and
indeed provide the strongest support yet for the crucial role played by cortisol in carbohydrate metabolism. Working with tilapia hepatocytes, one of us
in collaboration with A. Takemura (M. M. Vijayan
and A. Takemura, unpublished data) found that after
68 days of culture, the cells were no longer glycogenolytic, but synthesized glucose and were net producers of glycogen. Upon the addition of cortisol, the
tilapia cells consistently increased medium glucose in
a dose-dependent manner (101000 nM cortisol) over
a two-day period compared with the control. At the
same time, cortisol (100 and 1000 nM) also increased
glycogen breakdown compared with control, confirming cortisols glycogenolytic potential previously
reported in rat hepatocytes (Baqu et al., 1996). In
the presence of 3-mercaptopicolinate, an inhibitor of
gluconeogenesis, medium glucose dropped by 60%
without concurrent effects on glycogen content; it
therefore appears that cortisol-induced gluconeogensis can override glycogenolysis (M.M. Vijayan and
A. Takemura, unpublished data). Similarly, cortisol
uniformly increased medium glucose in primary cultures of rainbow trout hepatocytes, providing further
support for a gluconeogenic action of cortisol (M.M.
Vijayan, C. Pereira and G.K. Iwama, unpublished
data). It is likely that this increased gluconeogen-

esis provides C3 precursors for glycogen repletion as

suggested for mammals. Future studies are planned
to characterize the biochemical pathways triggered in
response to cortisol stimulation using the primary hepatocyte culture system. Our working hypothesis is that
cortisol exerts a nongenomic effect on glycogen metabolism and gluconeogenesis in fish, and characterizing
the signal transduction pathways for cortisol action
on carbohydrate metabolism should be a challenging
topic.
Very little is known concerning the effect of
cortisol on muscle glycogen content, and as with
liver glycogen, a clear trend as to the role of cortisol
on muscle glycogen remains elusive. Because of its
important role in muscle contraction and concomitant volatility, muscle glycogen is even less accessible
experimentally than liver glycogen. Further, quite
minor changes in metabolites, too small to be detected on a cell level, can have important repercussions
because of the bulk of the tissue. Cortisol treatment
in the Japanese eel (Anguilla japonica, Anguillidae)
increased muscle glycogen (Inui and Yokote, 1975), as
did cortisol replacement in hypophysectomized American eels (Butler, 1968). Similarly, muscle glycogen was significantly higher in dexamethasone-treated
rainbow trout (Pagnotta et al., 1994) than in shamtreated controls. In contrast, Lidman et al. (1979)
found a decrease in muscle glycogen one day after
cortisol injection, but not on day 4 or day 14 postinjection in the European eel, while muscle glycogen
content remained unaffected by cortisol treatment in
the roach (Rutilus rutilus, Cyprinidae) (Mller and
Hanke, 1974).
The decrease in muscle glycogen content associated with exercise can be abolished by pretreating rainbow trout with metyrapone, a blocker of
11-hydroxylation, suggesting a role for cortisol in
exercise-induced muscle glycogen mobilization (Eros
and Milligan, 1996). The higher lactate concentration associated with this glycogen breakdown may
be a source of aerobic fuel necessary to cope with
the increased energy demands of exhaustive exercise (Eros and Milligan, 1996), or if lactate reaches
the liver it may serve as a gluconeogenic precursor.
Although corticosteroid-dependent increases in blood
lactate are common in mammals, a study in the
European eel (Anguilla anguilla, Anguillidae) showed
that chronic cortisol treatment increased plasma lactate levels (Lidman et al., 1979) and blood lactate
increased acutely in New Zealand snapper (Pagrus
chrysophrys auratus, Sparidae) with corticosteroid

234
exposure (Bollard et al., 1993). Others, however,
failed to detect effects of chronic corticosteroid treatment on plasma lactate in brook charr (Vijayan et al.,
1991), rainbow trout (Andersen et al., 1991; Pagnotta
et al., 1994) and tilapia (Vijayan et al., 1997a). Certainly, additional research is warranted to understand
the peripheral action of cortisol in fish, including the
characterization of GR distribution. As lactate has
been shown to be an important substrate for gluconeogenesis in fish (Suarez and Mommsen, 1987),
and lactate gluconeogenesis is stimulated by cortisol
(Renaud and Moon, 1980; Janssens and Waterman,
1988; Mommsen et al., 1992), it is conceivable that the
mobilization of muscle glycogen and subsequent lactate production may be an important process providing
substrate for hepatic gluconeogenesis/glycogenesis.
Protein and amino acids
Protein turnover
General agreement seems to have developed that as
part of its widespread catabolic activity, cortisol exerts
a proteolytic action, especially on fish white muscle
and possibly in the liver (Barton et al., 1987). This
notion is based on numerous indirect observations
(mainly plasma amino acid concentrations; Vijayan
et al., 1997a), guided by the role of the hormone
in mammalian systems, although very little piscine
evidence has been put forth in direct support. First,
it is rather difficult to confirm a proteolytic action of
cortisol experimentally, unless protein and amino acid
turnover are assessed, which only one study has done
to date (Andersen et al., 1991). Second, considering
that white muscle makes up at least, if not more than,
50% of the weight of the fish, yet is characterized
by a slow metabolic rate, it is arduous to measure
changes, because metabolically important changes in
plasma or liver may remain well below the detection
limit in the muscle. Third, the relatively crude analysis
on fishes, usually done as part of experimentation with
other goals in mind, will preclude useful data, as will
shorter-term studies. As a result, only the exaggerated changes during extreme conditions help to point
at the role of cortisol in the regulation of fish muscle
proteolysis.
One of these situations is exposure to cortisol
during long-term starvation, which consistently leads
to weight loss greater than that in the absence of
cortisol (Storer, 1967; Pickford et al., 1970). Similar observations for reduced growth performance are
made for fish under natural or forced stress situ-

ations. Conversely, a precipitous drop in cortisol levels

in the starving snakehead (Ophiocephalus maculatus,
Channidae) is accompanied by reduced protein catabolism (Woo and Cheung, 1980). Another extreme
situation is the spawning migration of Pacific salmon species, which consumes a large percentage of
the white muscle and is characterized by exceptionally high titres of circulating glucocorticoid hormones
and high activity of the interrenal glands (McBride
et al., 1986). However, at the same time, the animals undergo additional drastic changes compounded
by starvation, including excessive exercise, hypoosmoregulation and sexual maturation. Any or all of
these can potentially affect muscle proteolysis, making the correlation with plasma cortisol somewhat
tenuous. Two additional natural hypercortisolaemic
conditions should be considered. The first, smoltification in salmonids, is correlated with drastically
increased cortisol levels (Specker and Schreck, 1982),
although experimentation has usually been focused on
gill function and other parameters and not on protein
or amino acid turnover. The other is exhaustive exercise, which is associated with elevated plasma cortisol
and altered plasma amino acid concentrations during
the recovery period (Milligan, 1997). In contrast, in
sustained aerobic exercise, the contribution of protein
and amino acids to oxidative pathways remains relatively low and unchanged (Lauff and Wood, 1996;
Kieffer et al., 1998). One wonders whether the discrepancy to a previous set of experiments (Van den
Thillart, 1986), which reported high endogenous rates
of amino acid oxidation, might not result from the
use of freshly cannulated (stressed?) and thus hypercortisolaemic trout in the older study. At any rate,
plasma amino acids tend to be increased in fish with
cortisol implants (Andersen et al., 1991; Vijayan et
al., 1997a). Together, these observations strongly suggest a role for cortisol in peripheral proteolysis in
fishes. As shown below, this role may be linked to
substantial readjustments in amino acid metabolism,
affecting deamination, transamination, ammonia production and flux of amino acid carbons into glycogen,
gluconeogenesis or proteins under the influence of the
hormone.
In addition to these peripheral actions, cortisol may
also have some influence on liver protein. While in
brook trout liver size was unaffected (Vijayan et al.,
1991), in coho salmon cortisol treatment decreased
the hepatosomatic index without imbalancing hepatic
protein output, as no changes in the overall plasma
proteins levels were detected (Vijayan and Leather-

235
land, 1989). The overall contribution of liver protein to
the amino acid pools and amino acid turnover is likely
to be modest, considering the relatively small size of
this organ. It should not be ignored, however, that
oestradiol, another steroid hormone, can affect liver
size by hyperplasia and hypertrophy (Korsgaard and
Mommsen, 1993). An interesting relationship between
cortisol and oestradiol on protein metabolism can be
envisaged in vitellogenic females or even in feminized
males (Sumpter and Jobling, 1995). In other species,
cortisol was also proteolytic, and led to decreases in
the hepatosomatic index (Barton et al., 1987). This
catabolic effect was especially pronounced in fasting
specimens. In contrast, the liver of the Japanese eel
responds to large doses of cortisol with an increase in
size, resulting in a substantially increased hepatosomatic index, in spite of the fact that the animals were
fasting for about 10 days (Inui and Yokote, 1975).
However, the eel did show the commonly observed
increase in aspartate aminotransferase, concomitant
with increases in fructose 1,6-bisphosphatase, and an
unexpected, highly significant, increase in the activity
of phosphofructokinase-1 (Inui and Yokote, 1975). It
appears that gluconeogenesis and glycogenolysis are
increased simultaneously, likely resulting in a higher
degree of futile cycling at this key metabolic control point. While this is prone to lend more control
power to this specific site, it might contribute to the
increased metabolic demand, i.e. oxygen consumption
observed in the presence of pharmacological doses of
glucocorticoid. In conjunction with these key metabolic changes, cortisol causes a slight degranulation
of the -cells in the eel endocrine pancreas (Inui and
Yokote, 1975), suggesting increased activity of the cells under these conditions. The potential interplay of
insulin with glucocorticoid is discussed elsewhere in
this review (see p. 250).
Conversely, cortisol decreases growth rates in
feeding fish (Davis et al., 1985; Barton et al., 1987;
Pickering, 1990); without additional experimentation,
however, it is impossible to judge whether increased
proteolysis or reduced rates of protein synthesis, or
both, are at the root of this phenomenon. In the fetal
sheep model, cortisol increased proteolysis, without
affecting the rate of protein synthesis from leucine
(Milley, 1995). Glucocorticoids are known to retard
tissue growth and to inhibit proliferation in cells of
hepatic and nonhepatic origin. For instance, as in
rat liver, dexamethasone administration will decrease
the rate of thymidine incorporation into DNA in trout
fibroblasts (Lee and Bols, 1989a, 1989b; Van Oostrom

and Bols, 1991), decrease the growth rate in a rainbow

trout gonadal cell line (Lee et al., 1986) and change
cell adherence, similar to fibronectin synthesis induction in rat liver cells. These effects were achieved with
cortisol, similar to fibronectin synthesis induction in
rat liver cells, but not with corticosterone. The situation is slightly different for cultured channel catfish
(Ictalurus punctatus, Ictaluridae) hepatocytes, where
both corticosterone and dexamethasone decreased the
rate of 3 H-leucine incorporation into protein (Prosser
et al., 1991). Other options to explain the compromised growth of fish is that cortisol interferes at the
neural level with satiation, intraspecies aggression or
with nutrient uptake across the intestine. Cortisol is
one of the hormones modulating amino acid transport
in the intestine. Smolting coho salmon, for instance,
will show stimulated L-proline uptake when implanted
with cortisol for two weeks, with the hormone significantly altering Km as well as Vmax of proline uptake
(Collie and Stevens, 1985). Unfortunately, it is not
clear whether this effect is related to the ontogentic
stage of the fish (smoltification), which happens to be
correlated with increased cortisol titres (Specker and
Schreck, 1982), or whether it constitutes a more general function of the hormone. It is also possible that
exposure to exogenous cortisol affects the efficiency
of intestinal nutrient uptake by exerting a detrimental
influence on the integrity of the intestinal lining, thus
adversely affecting growth (Barton et al., 1987). In
the context of nutrient reabsorption, cortisol may act
at different levels, through a combination of indirect
and direct effects. In two studies showing decreases in
fish growth, cortisol was added directly to the food,
yet cortisol in food is likely to affect the gut lining,
decreasing nutrient availability (food reabsorption,
protein digestibility and food conversion efficiency;
Barton et al., 1987). Therefore, impaired growth may
be simply due to direct effects of cortisol on nutrient
availability to the fish. However, coinciding with loss
in body weight in cortisol-implanted rainbow trout is
a depression of plasma triiodothyronine (T3, but not
T4) (Brown et al., 1991), an observation confirmed
for coho salmon (Vijayan and Leatherland, 1989) and
European eel (Redding et al., 1986). It is accepted that
T3 constitutes one of the key regulators of nutrient
uptake in fishes in anabolic states and is positively
correlated with body mass, glucose and amino acid
transport, Na+ /K+ -ATPase and glucose permeability
of the intestine (Reshkin and Ahearn, 1987; Collie and Ferraris, 1995). Hence, a cortisol-dependent
decrease in plasma T3, most likely brought about by

236
changes in 50 -deiodinase by the steroid (Vijayan et al.,
1988; Brown et al., 1991), will indirectly decrease
nutrient absorption from the intestine. The interaction
between T3 and cortisol may not end here. Prosser and
co-workers reported sizable decreases in hepatic protein synthesis with T3 in cultured catfish hepatocytes
(Prosser et al., 1991). If we accept that cortisol exerts
a negative influence on plasma T3 levels, the steroid
may release a T3 brake on hepatic protein synthesis.
The abundance of fish muscle means it has an
enormous potential to contribute amino acids derived
via proteolysis to catabolic pathways. In extreme
cases, such as spawning migration of salmonids and
other fishes, major portions of the migration appear
to be fuelled preferentially by amino acids; in resting fish, protein utilization may account for only
a small proportion of oxygen consumption, a value
that increases during sustained swimming but never
exceeds 3040% of nutrient use (Lauff and Wood,
1996). In the migrating salmon, hypertrophy of interrenal tissue is observed and circulating concentrations
of cortisol exceed normal levels. While the potential causality between hypercortisolaemia and muscle
proteolysis has only been analysed in mammals and
needs to be established for the fishes, the profiles of
amino acids leaving the muscle differ dramatically
between mammals and fishes. In the mammals, alanine and glutamine make up the overwhelming bulk
of amino acids transporting carbons and nitrogen to
other tissues, preferentially the liver. This metabolic
scenario is reflected in high activities of glutamine
synthetase and aminotransferases in muscle. Although
the data sets available for fishes are fairly limited, an
entirely different picture arises: alanine is the main
nitrogen carrier leaving the muscle, while glutamine
plays an ancillary role at best. This is confirmed by
minute levels of glutamine synthetase in fish white
muscle, juxtaposed to the activity of glutaminase, and
glutamine constitutes a preferred oxidative substrate
for red muscle mitochondria (Chamberlin et al., 1991;
Chamberlin and Ballantyne, 1992).
In conjunction with rates of protein synthesis in
muscle lower than controls, the stepped-up rate of
peripheral proteolysis will generate an increased availability of amino acids to the system. However, if a
given amino acid enters and subsequently leaves the
plasma pool at an exaggerated rate. This will not
inevitably be reflected in alterations to the plasma
concentration. In the migrating salmon, for instance,
with its rampant peripheral proteolysis, only very few
plasma amino acids reveal changes in their static con-

centrations (Mommsen et al., 1980), while amino acid

turnover can be calculated to be increased substantially (French et al., 1983). In fact, the main conclusion
of one of these studies (Mommsen et al., 1980) was
that at least for the migrating salmon only those
amino acids that are not easily converted into usable
carbon backbone will undergo concentration changes
in plasma, simply because their metabolism is slow
or targets are few. Interestingly, those amino acids
experiencing large changes in turnover, most likely
alanine and glutamate, incur only minor fluctuations
in plasma concentration. Likewise, intracellular amino
acid concentrations do not change much in muscle or
liver, even after infusion of amino acids or a highprotein meal that floods the plasma with free amino
acids, implying that these tissues have a high capacity
to regulate flux without affecting stationary concentrations. Therefore, it is exceedingly difficult to estimate
flux direction or speed for any given amino acid,
unless compartment uptake and traffic are known.
Branched-chain amino acids, for instance, are preferentially oxidized by the muscle (with high titres
of the corresponding aminotransferase; Moon, 1983)
while glutamate, alanine and glutamine are most likely
destined for the liver.
Even the ultimate fate of the amino acids or their
carbons is under debate, with gluconeogenesis, oxidation and protein synthesis as suitable candidates. The
view that protein mobilization provides amino acids
for hepatic gluconeogenesis has not found universal
experimental support, because in some studies, nonprotein sources have been indicated as predominant
gluconeogenic substrates in the presence of cortisol.
The estimate for isolated trout hepatocytes that around
80% of oxygen consumption is allocated to the synthesis of protein (Houlihan et al., 1995) leaves one
wondering how cortisol, with its increased oxygen
consumption and ammonia output from the fish, may
influence hepatic protein synthesis. If indeed the hormone causes hepatic proteolysis, even higher proportions of oxygen consumption will be available to be
allocated to other pathways.
Amino acid metabolism
The increased peripheral proteolytic activity seems to
be accompanied by substantial adjustments in amino
acid metabolism, including ammonia output by the
fish, activities of transaminases and numerous other
enzymes involved in or ancillary to amino acid metabolism in the liver. Cortisol plays an important role in
the control of all these parameters, even if it has not

237
always been possible to detect changes in amino acid
metabolism based on snapshot views of their plasma
concentrations. A summary of the proposed cortisoldependent cascade involving amino acids and their
metabolism is presented in Figure 4 (p. 246).
Different experimental approaches have been used
to analyse alterations in amino acid metabolism in
conjunction with cortisol. These range from correlating daily endogenous cortisol rhythms with metabolic
parameters, through less subtle, yet still endogenous, hormone changes brought about by exhaustive
exercise, stressful conditions such as crowding, or
SW adaptation, to pharmacological treatment of fish
or cells with cortisol or cortisol agonists. In most
instances, tissue amino acid mobilization and amino
acid levels in plasma tend to be positively correlated
with cortisol titres.
Ammonia output. Under the influence of cortisol,
plasma amino acids increase and are subsequently
funnelled into key metabolic pathways, including
glycogenesis, gluconeogenesis and possibly protein
synthesis. The first two involve transdeamination
and hence may result in increased ammonia output,
although, as seen below, the site of deamination is not
entirely clear. All three pathways coincide with, if not
cause, increases in oxygen uptake. The most interesting aspect of this metabolic adjustment is that only
very small changes in plasma cortisol are required to
alter amino acid metabolism. In resting and unstressed
toadfish, for instance, at low endogenous cortisol concentrations, ammonia excretion scales with plasma
cortisol (Figure 2A). While this correlation does not
necessarily reflect causality, it holds only over a fairly
narrow range and, most importantly, at low concentrations of the steroid. As soon as a low threshold is
crossed, be it through experimental manipulation or
natural crowding stress, the correlation breaks down
and cortisol concentration and ammonia excretion or
total nitrogen excretion appear as independent entities (Figure 2B), and other effects attributed to the
hormone are noted (see below). Of course, it should
be kept in mind that especially concerning nitrogen
excretion, the facultative ureogenic toadfish may not
constitute the best model for teleost fishes in general.
However, other models support the above contention. Starvation in the snakehead (Woo and Cheung,
1980), an air-breathing and potentially ureogenic species, and in other more ordinary teleosts such as the
goldfish (Carassius auratus, Cyprinidae), lake trout
(Salvelinus namaycush, Salmonidae) or sea raven,

Figure 2. Correlation between plasma cortisol level and total nitrogen excretion in Gulf toadfish (Opsanus beta). (A) Undisturbed
(control) fish. Regression is significant at p < 0.001; y = 0.0386x
+ 0.88, r2 = 0.80. (B) Crowded/confined (chronically stressed) fish;
p < 0.94; y = 0.003x + 3.645, r2 = 0.001. Lines indicate least-squares
regression. Recalculated and redrawn from Hopkins et al. (1995).

238
causes decreases in plasma cortisol levels, accompanied by reduced ammonia excretion. We therefore
conclude that at physiological concentrations, cortisol
may play a housekeeping role in partitioning of amino
acid carbon into nonprotein pathways.
With regard to protein synthesis, clearly additional experimentation seems warranted. When cultured tilapia hepatocytes are exposed to cortisol, the
rate of glucose production through gluconeogenesis
more than doubles (M.M. Vijayan and A. Takemura,
unpublished data), but the rate of ammonia release
from the cells appears to be unchanged (M.M. Vijayan
and T.P. Mommsen, unpublished data). While these
results may suggest nonprotein sources for the additional glucose, we would like to caution that the use of
common culture medium, such as L-15 that contains
high pyruvate and galactose, may bias cell metabolism
in favour of carbohydrate sources. In isolated catfish
hepatocytes, for instance, the simultaneous presence
of lactate and alanine eliminates the high production
rate of ammonia observed with alanine alone (Casey et
al., 1983). Clearly, at this point there is enough evidence to suggest that both gluconeogenesis and amino
acid turnover are activated by cortisol, with the outcome of individual experiments partially governed by
the imposed metabolic condition. Although such isolated systems are ideal to identify potential metabolic
targets for cortisol, care must be taken not to over
interpret such data. Obviously, we cannot make any
statements about amino acid gluconeogenesis until
we have simulated more physiological conditions for
the isolated liver cells. This scenario would include
low concentrations of pyruvate, possibly substantial
concentrations of lactate to simulate postexercise conditions, absence of galactose and increased levels
of exogenous amino acids, compared with standard
cell culture media, usually developed specifically for
mammalian cells.
Enzymes
The increased rates of ammonia output coincide with
key adjustments in amino acid metabolism, especially
in the enzymes involving the processing of glutamate, i.e. glutamine synthetase, aminotransferases and
glutamate dehydrogenase.
Glutamine synthetase. In the toadfish, confinement/crowding (Walsh and Milligan, 1995) or injection with dexamethasone (Mommsen et al., 1992)
will induce hepatic glutamine synthetase, the unique
feeder enzyme controlling nitrogen entry into the

urea cycle in this ureogenic species. The toadfish

respond to the imposed stress with a transient plasma
cortisol surge, from about 10 to 37 ng mL1 , peaking after 2 h and subsiding within about 24 h.
This surge is followed 24 h later by a substantial increase in the activity of glutamine synthetase
(Table 6, p. 241). The induction by cortisol seems
to be specifically directed towards the cytosolic version of the enzyme, while the mitochondrial form
remains unaffected (Walsh and Milligan, 1995). Preinjection of toadfish with metyrapone prevents these
stress-dependent increases in cortisol and forestalls
any increases in hepatic glutamine synthetase activity
(Hopkins et al., 1995), providing convincing evidence that the enzyme increase is directly related to the
preceding cortisol surge. Because fish liver glutamine
synthetase does not seem to be activated allosterically
(Shankar and Anderson, 1985), cortisol must be regulating transcription of the glutamine synthetase gene.
The interesting points here are that a relatively small
surge in endogenous cortisol precedes the increases in
enzyme activity and that plasma cortisol has returned
to baseline by the time increases in enzyme activity
are noted. The susceptibility of glutamine synthetase
to induction by corticosteroids is potentially a general
phenomenon and not necessarily related to urea synthesis. A doubling of glutamine synthetase activity had
been observed not only in toadfish, which in retrospect
belonged in the crowded/stressed category, injected
with dexamethasone, but also in sea raven exposed
to cortisol (Vijayan et al., 1996a). In the context of
urea cycle regulation by corticosteroids, the toadfish
shows no increases in hepatic urea output and does
not follow the established mammalian pattern, which
is characterized by increased activity and expression
of numerous urea cycle enzymes (Christowitz et al.,
1981; Husson et al., 1990).
This induction of glutamine synthetase is not
restricted to the liver, as glucocorticoids induce
glutamine synthetase expression in a number of mammalian tissues, including glial, retinal and muscle
cells (Kumar et al., 1986). To date, similar phenomena have not been analysed in fish systems, but it is
well known that ammonia stress leads to the accumulation of glutamine and concurrent depletion of
glutamate in the goldfish brain (Levi et al., 1974).
Therefore, we can hypothesize that this is not simply
due to mass-action effects, but that, under the influence of glucocorticoids, brain glutamine metabolism
will reach a new steady state, likely involving de novo
synthesis of glutamine synthetase. Not surprisingly,

239
the mammalian and avian glutamine synthetase genes
contain upstream glucocorticoid-response elements
(Zhang and Young, 1991; Fahrner et al., 1993); based
on the limited experimental evidence about enzyme
induction in fishes, we can predict the presence of
such elements for the piscine glutamine synthetase
gene(s).
Exhaustive exercise in trout poses a situation with
elevated plasma cortisol (Milligan, 1997), where
the rate of ammonia production, largely through
the purine nucleotide cycle in muscle (Mommsen
and Hochachka, 1988), overwhelms excretory rates.
As a result, plasma ammonia increases dramatically (Mommsen and Hochachka, 1988). Considering
the induction of glutamine synthetase in mammalian
tissues, it is likely that fish display a similar phenomenon, again driven by cortisol and thus contributing to the protection of neural tissue from the effects of
increased plasma ammonia. In addition, mammalian
muscle glutamine synthetase is induced by glucocorticoids (Hickson et al., 1996), but fish muscle contains
only small amounts of the enzyme compared with
mammalian muscle (Chamberlin et al., 1991), making an important physiological role for the enzyme
questionable in the absence of massive activation by
glucocorticoids.
Aminotransferases. Two important lessons have
emerged from experiments with toadfish showing
a direct relationship between ammonia and plasma
cortisol and delayed response of an enzyme to a
preceding cortisol surge. One is that low concentrations of cortisol can regulate metabolism, and
the other that cortisol changes may be initiated
slowly, but are designed for longer-term action. These
considerations are relevant, as a number of processes can influence plasma cortisol levels and may
thus potentially alter metabolic baselines. In some
experiments, plasma cortisol concentrations undergo
substantial diurnal cycles (Davis et al., 1984), at
times with specimen-specific rhythms (Gomez et al.,
1996), and in threespine sticklebacks (Gasterosteus
aculeatus, Gasterosteidae) that fail to show diurnal
cortisol cycles, shorter periods of daylight significantly lowered plasma cortisol concentrations compared with fish maintained under longer daylight conditions. This effect was especially pronounced in
males (Audet et al., 1986). However, the potential
influence of endogenous variations in corticosteroids
on a daily or seasonal basis or of those associated
with preovulation (Jalabert, 1976) and differences

between the sexes (Miranda et al., 1992) on intermediary metabolism are as yet undefined. A discussion
of potential effects is therefore deemed premature. In
other experiments, feeding events may be followed by
a minor peak in plasma cortisol. In unstressed rainbow
trout, this surge reaches levels about fourfold higher
than prefeeding, and within 4 h, plasma cortisol has
attained resting levels (Bry, 1982). As shown in the
toadfish, such a transient surge is sufficient to readjust
metabolic output. One wonders whether postprandial
ammonia and oxygen uptake surges or those following
infusion with amino acids observed in catfish (Brown
and Cameron, 1991), may in part be due to spikes in
endogenous plasma cortisol. In the catfish, both situations are accompanied by large increases in amino
acid turnover usually the signature of cortisol action.
Although these intrinsic fluctuations may appear small
compared with acute or chronic stress situations metabolically they seem to affect baselines on which other
effects are superimposed, or conversely other effects
may be masked by the experimental noise raised in
response to cortisol. This altered baseline may explain
in part the controversy that has arisen from seemingly
similar experimental approaches resulting in opposing
metabolic actions.
For instance, the induction of hepatic tyrosine
aminotransferase (TAT), the enzyme controlling the
degradation of tyrosine and a prime model for induction and glucocorticoid-dependent gene regulation in
mammals (Collier et al., 1996) and the structure of
mammalian GREs, has been found to respond in a
seemingly unpredictable way in piscine systems. On
the one hand are studies that fail to find corticosteroiddependent increases in enzyme activity in white bass
(Morone chrysops, Percichthyidae), black crappie
(Pomoxis nigromaculatus, Centrarchidae), two oncorhynchids and toadfish; on the other hand are reports
showing unequivocal inducibility of the enzyme by
cortisol or dexamethasone. Species included in this
group are the brook trout, rainbow trout, channel
catfish, and sea raven (Table 5, p. 240).
Although the brook trout falls into the group
of positive responders, an interesting aspect of the
study by Whiting and Wiggs (1977) is that a paired
re-analysis of their data by us reveals that a single
saline injection is sufficient to significantly increase
the hepatic levels of TAT. This increase is noted 4
days after the injection, compared with the noninjected control fish. One wonders whether the expected
single peak in endogenous cortisol caused by the handling stress in the saline-injected fish may be correlated

240
Table 5. Effects of corticosteroids on tyrosine aminotransferase (TAT) activity in liver tissue or isolated liver cells of teleostean fishes
Species
Rainbow trout
(Oncorhynchus mykiss)

Brook charr
(Salvelinus fontinalis)
Sea raven
(Hemitripterus americanus)
Channel catfish
(Ictalurus punctatus)
Coho salmon
(Oncorhynchus kisutch)
Chinook salmon
(Oncorhynchus tshawytscha)
White bass
(Morone chrysops)
Black crappie
(Pomoxis nigromaculatus)
Gulf toadfish
(Opsanus beta)

System

Agonist

Exposure

Tissue culture
Cultured hepatocytes

Cortisol
Cortisol
Dexamethasone

I.m. injection

Cortisol

14 d

I.p. injection

Cortisol

5d

1.9 fold

Vijayan et al. (1996a)

In feed

Cortisol

10 wk

1.9 fold

Davis et al. (1985)

I.p. injection

Triamcinolone

4 h3 d

No change

Fellman et al. (1971)

I.p. injection

Triamcinolone

4h

No change

Fellman et al. (1971)

I.p. injection

Cortisol

5d

No change

Chan and Cohen (1964)

I.p. injection

Cortisol

5d

No change

Chan and Cohen (1964)

I.p. injection

Dexamethasone

3d

No change

Mommsen et al. (1992)

5h
5d
5d

Increase

Reference

2 fold
1.3 fold
1.7 fold

Lipsky et al. (1986)

Devaux et al. (1992)
Devaux et al. (1992)

1.8 fold (max.) Whiting and Wiggs (1977)

Increase was measured against a control exposed to insulin and dexamethasone.

I.m. intramucscular; I.p. intraperitoneal.

with the observed increase in liver TAT. By the same

token, the scope of activation by cortisol is artificially
diminished owing to the positive response of the shaminjected group. A similar trend, unfortunately not
analysed statistically, is visible in an earlier study on
the effects of cortisol on rainbow trout, where activity
of aspartate aminotransferase in the liver appeared to
have been higher in fish simply given daily sham injections compared with untreated control fish. The effects
of pharmacological doses of cortisol were then superimposed on handling-dependent altered background
activity (Freeman and Idler, 1973). In summary, it
may not be a coincidence that all studies reporting
resilience of TAT to cortisol treatment used intraperitoneal injection of the hormone, often on a daily basis.
Refining application techniques for the hormone and
higher resolution of induction of the enzyme through
molecular methods may alter our view of the relationship between cortisol and TAT in fishes, as would
the localization of a conserved GRE in the upstream
region of the piscine TAT gene.
Pursuing this line of thought further, other
examples show that the hormone injection technique
may lead to an erroneous interpretation of the effectiveness of the hormone. Leach and Taylor (1982)

injected cortisol on a daily basis into fasting mummichog (Fundulus heteroclitus, Cyprinodontidae) and
noticed no differences in liver amino acid concentrations compared with fish injected with vehicle (peanut
oil) only. However, both groups showed significantly higher levels than uninjected animals. Again,
it is likely that a cortisol peak brought about by the
daily handling stress was sufficient to cause proteolysis and subsequent increases in liver amino acid
titres. Together, these data not only reiterate that
handling forms a critical feature of the experimental
protocol, but also point to the potential of small transient increases in plasma cortisol to substantially alter
fish metabolism, ranging from proteolysis, through
amino acid levels to enzyme induction. Such small,
longer-term or delayed effects may help to define
cortisols elusive housekeeping functions. At the same
it poses the question whether pharmacological doses
of cortisol do not provide a distorted picture of the hormones targets and effectiveness, and that differences
in enzyme responses may not be rooted in different
kinetics of gene expression, mRNA stability and/or
protein turnover for a given enzyme. As mammalian
models have clearly shown, all these parameters are
potentially regulated by cortisol.

241
Table 6. Efects of corticosteroids on activity of enzymes involved in amino acid metabolism in liver
tissue or isolated liver cells of teleost fishes
Enzymes and species

Agonist

Increase

Reference

Carassius auratus
Hemitripterus americanus
Oncorhynchus mykiss
Salvelinus fontinalis

Cortisol
Cortisol
Stress
Cortisol

Anguilla japonica
Fundulus heteroclitus
Opsanus beta
Pleuronectes platessa

Cortisone
Cortisol
Cortisol
Dexamethasone
Cortisol

2.4 fold
1.5 fold
> 2 fold
1.4 fold
No change
1.5 fold
No change
No change
No change
No change

Storer (1967)
Vijayan et al. (1996a)
Morales et al. (1990)
Freeman and Idler (1973)
Tam et al. (1988)
Freeman and Idler (1973)
Inui and Yokote (1975)
Leach and Taylor (1982)
Mommsen et al. (1992)
White and Fletcher (1985)

Chan and Woo (1978)

Inui and Yokote (1975)
Vijayan et al. (1996a)
Freeman and Idler (1973)
Mommsen et al. (1992)

Alanine aminotransferase

Aspartate aminotransferase
Anguilla japonica

Cortisol

Hemitripterus americanus
Oncorhynchus mykiss
Opsanus beta

Cortisol
Cortisol
Dexamethasone

1.5 fold
1.6 fold
1.6 fold
1.4 fold
1.5 fold

Cortisol
Stress
Dexamethasone

1.4 fold
1.5 fold
1.6 fold

Vijayan et al. (1996a)

Morales et al. (1990)
Mommsen et al. (1992)

Cortisol
Dexamethasone
Crowding/confinement

2.1 fold
2 fold
4 fold

Vijayan et al. (1996a)

Mommsen et al. (1992)
Walsh and Milligan (1995)

Glutamate dehydrogenase
Hemitripterus americanus
Oncorhynchus mykiss
Opsanus beta
Glutamine synthetase
Hemitripterus americanus
Opsanus beta

Enzyme increase is noted in acid-exposed fish that go through phases of hypercortisolaemia.

While at present TAT may not constitute the same

powerful experimental tool as in mammalian liver,
the enzyme is clearly inducible; therefore, at least
in responsive species, the enzyme could be useful
in studying corticoid action in fishes. Its activity or
mRNA could serve as tools to determine whether
enzyme abundance is increased owing to nuclear
actions of the corticosteriod involving de novo synthesis or via permissive effects enrolling other activation mechanisms. However, such activation is not
restricted to TAT, but also applies to the ubiquitous
alanine and aspartate aminotransferases and glutamate dehydrogenase, the key enzyme in mitochondrial
deamination of amino acids (Table 6). It would be
interesting to look at compartment-specific induction
of alanine and aspartate aminotransferases, which both
exist in cytosolic and mitochondrial isozymes in fish

liver. Against the backdrop of the induction of the

cytosolic isozyme of glutamine synthetase in the toadfish and the exclusively mitchondrial glutamate dehydrogenase in a few species, it seems likely that glucocorticoids exert a generalized, and not compartmentselective, positive effect on hepatic enzymes involved
in amino acid metabolism.
The fact that higher titres of alanine aminotransferase and glutamate dehydrogenase could potentially
increase the flux of alanine through the transdeamination route is confirmed for isolated sea raven and
trout hepatocytes. Cortisol enhances alanine oxidation (Vijayan et al., 1993b) and alanine gluconeogenesis (Vijayan et al., 1994b) and, by implication, the
rate of ammonia production. Finally, in rat hepatocytes, cortisol and synthetic glucocorticoids stimulate
the transport of neutral amino acids (Le Cam and

242

Table 7. Proteins induced by glucocorticoids in teleostean fishes

Agonist

Tissue / Species

Glucose 6-phosphatase
PEPCK
F1,6BPase

C
C
C

Liver, eel
Liver
Liver

GAPDH
PFK-1
Allantoicase
Arginase
Malate dehydrogenase
-Glycerophosphate DH
Malic enzyme
Glucose 6-phosphate DH

C
C
C
C
D
C
C
C

Succinate dehydrogenase
NADPH cytochrome C reductase
HOAD

C
C
C
C
C
C
D

References

Enzymes

Glycerol kinase
CYP1A1
P4503A
EROD
Superoxide dismutase
Catalase
Na+ /K+ -ATPase
Ca2+ -ATPase
H+ -ATPase
Calcium pump
Deiodinase
Triacylglycerol lipase

D/C
D
D
C
C
C
C
C
C
C

Inui and Yokote (1975), Chan and Woo (1978)

Foster and Moon (1986), Vijayan et al. (1996a, 1997a)
Inui and Yokote (1975), Chan and Woo (1978),
Vijayan et al. (1991)
Liver
Vijayan et al. (1991)
Liver
Inui and Yokote (1975)
Liver
Vijayan et al. (1996a)
Liver
Vijayan et al. (1996a)
Liver
Mommsen et al. (1992)
Liver
Vijayan et al. (1991)
Hepatocytes
Vijayan and Mommsen (unpubl.)
Liver
Nagayama et al. (1973), Morales et al. (1990),
Vijayan et al. (1991)
Gill
Langdon et al. (1984)
Liver microsomes
Hansson and Lidman (1978)
Charr liver
Vijayan et al. (1991)
Sea raven liver
Vijayan et al. (1996a)
Liver
Vijayan et al. (1991)
Liver cell line
Celander et al. (1996)
Liver cells
Devaux et al. (1992)
Devaux et al. (1992); Celander et al. (1996)
Liver, in the presence of -NF Devaux et al. (1992)
Erythrocytes, liver
ikic et al. (1986)
Liver
ikic et al. (1986)
Gill
McCormick (1990), Madsen et al. (1995)
Gill
Flik and Perry (1989)
Gill
Lin and Randall (1993)
Gill
Flik and Perry (1989)
Liver
Vijayan et al. (1988), Brown et al. (1991)
Liver, red muscle
Sheridan (1986)
Mesenteric lipid
Sheridan (1986)

D
C
C
C
C and stress
C
C
C

Plasma
Cultured hepatocytes
Gill/opercular membrane
Liver
Plasma
Plasma
Liver
Plasma

Others
Haemoglobin
Metallothionein
Chloride cells
Vitellogenin mRNA
Plasma protein
Oestradiol binding capacity
Hepatic soluble protein
C-reactive protein

ikic et al. (1986)

Hyllner et al. (1989)
Laurent and Perry (1990), McCormick (1990, 1995)
Ding et al. (1994)
Smith and Thomas (1991)
Smith and Thomas (1991)
Murat et al. (1981)
White and Fletcher (1985)

Abbreviations (see also on page 212): C cortisol; D dexamethasone; DH dehydrogenase; -NF -Naphthoflavone; EROD 7ethoxyresorufin-O-deethylase; GAPDH glyceraldehyde 3-phosphate dehydrogenase; HOAD hydroxyacyl-coenzyme A dehydrogenase;
NADPH nicotinamide adenine dinucleotide phosphate; PFK-1 phosphofructose kinase-1.
No effects of dexamethasone in Gulf toadfish (Mommsen et al. (1992) or cortisol in Mozambique tilapia (Vijayan and Mommsen, unpubl.).
No effects of cortisol in flounder (Pleuronectes platessa, Pleuronectidae) (Overnell et al., 1987).

243
Table 8. Parameters negatively affected by cortisol (C) or dexamethasone (D) in teleost fishes
Agonist

Tissue

References

D
D

Liver cells
Liver cells

Dasmahapatra and Lee (1993)

Dasmahapatra and Lee (1993)

C
C
C
C
C
C
C
C
C
C
C
C
C and stress
C and stress

Trout liver cells

Liver
Liver
Gill
Liver
Hepatocyte
Plasma
Plasma
Cartilage
Red blood cells
Pituitary
Plasma
Macrophages
Plasma

M.M. Vijayan et al. (unpublished)

Smith and Thomas (1991)
Valotaire et al. (1993)
Weisbart et al. (1987)
Pottinger (1990)
M.M. Vijayan and A. Takemura (unpublished)
Ding el al. (1994)
Carragher et al. (1989)
Takagi and Bjrnsson (1997)
Bollard et al. (1993)
Carragher et al. (1993)
Carragher et al. (1989)
Kaattari and Tripp (1987)
Pickering (1984)

Enzymes
EROD
CYP1A1
Others
Heat shock protein 70
Oestradiol binding capacity
Oestrogen receptor mRNA
Cortisol binding capacity
Vitellogenin

Glycosaminoglycan
Mean cellular Hb content
Gonadotrophin
Lymphokines
Lymphocytes
For abbreviations see Table 7.

Freychet, 1977); if the same holds true for fish liver,

this process will further enhance the effects of cortisol
on amino acid turnover and on gluconeogenic flux.
Arginase. One other enzyme involved in amino acid
turnover and potentially targeted by glucocorticoids is
liver arginase, a predominantly mitochondrial enzyme
in fishes (Mommsen and Walsh, 1989, 1991). In
cortisol-treated sea ravens, the activity of the enzyme
is increased twofold over vehicle-injected controls
(Vijayan et al., 1996a). It is open to discussion whether
this increase may be related to enhanced activity of
the urea cycle in this presumed largely ammoniotelic
species or to increased peripheral proteolysis. This
particular response is not without precedent in mammals (Kumar and Kalyankar, 1984; Jenkinson et al.,
1996) where cytosolic liver arginase is induced by
high protein intake or starvation, i.e. in periods of
increased ammonia turnover and where the arginase
gene contains sites binding with members of the
CCAAT/enhancer binding protein family that are regulated by glucocorticoid (Gotoh et al., 1997). Fish
liver arginase in general seems to be activated under
conditions of increased peripheral proteolytic activity, e.g. starvation (Grubinko et al., 1988; Singh and
Singh, 1988), which, as noted above, are normally
associated with decreasing concentrations of plasma

cortisol. Interestingly, arginase activity was not altered

by dexamethasone alone or in combination with glucagon in the ureagenic toadfish (Mommsen et al.,
1992).
Other effects
In addition to those enzymes directly involved in
amino acid metabolism, activities of a number
of ancillary enzymes are also positively influenced
by cortisol or dexamethasone. The list (Table 7)
includes key enzymes of gluconeogenesis (PEPCK,
fructose 1,6-bisphosphatase, glucose 6-phosphatase),
glycolysis (phosphofructose kinase-1, glyceraldehyde 3-phosphate dehydrogenase), hydrogen shuttles
(-glycerophosphate dehydrogenase, malate dehydrogenase), pentose shunt (glucose 6-phosphate
dehydrogenase), mitochondrial oxidative metabolism
(hydroxyacyl-coenzyme A dehydrogenase (HOAD),
succinate dehydrogenase, cytochrome C oxidase),
transport (ATPases, pumps) and detoxification (cytochrome P450s, superoxide dismutase, catalase). A
few proteins and ancillary parameters are negatively affected by these glucocorticoids (Table 8).
While Vmax measurements and the experimentally convenient direction of measurement fail to
furnish much information about actual metabolic
flux, glucocorticoid-dependent increases in these

244
key enzymes, together with a plethora of additional information on proteolysis, hyperglycaemia
etc., allows us to synthesize possible shifts in metabolic preferences under the influence of glucocorticoids.
Although cortisol is considered to be largely catabolic in scope, the hormone induces the synthesis of
a number of proteins that from a metabolic point of
view seem initially counterintuitive. However, to be
able to utilize the compounds made available by proteolysis effectively, the metabolic machinery must be
at hand. Not in all cases can metabolic flux through
key pathways be controlled by allosteric mechanisms
or through mass action ratios and hence it is not
surprising that glucocorticoids induce a number of
hepatic enzymes. These increases in enzyme titres are
caused by de novo biosynthesis of additional enzyme
molecules because corticosteroid-dependent increases
can be blocked by the concomitant exposure of the
experimental system to protein synthesis inhibitors
such as actinomycin D (Ignatius and Oommen, 1990),
thus ruling out decreased enzyme degradation as the
hormonal target.
The data collated in Table 7 imply that the overall
rate of protein synthesis is increased in fish liver under
the influence of glucocorticoids, with a bias towards
enzymes involved in amino acid turnover. This activation is not biased towards certain compartments, as
enzymes found in the mitochondria (e.g. cytochrome
C oxidase, glutamate dehydrogenase), cytosol (e.g.
PFK-1, GAPDH, lactate dehydrogenase), microsomes
(e.g. glucose 6-phosphatase, detoxification enzymes)
and plasma membranes (e.g. ATPase) are all represented. Together with the alleged peripheral proteolysis
and resulting higher plasma amino acids, and backed
by increased activities of glutamate dehydrogenase,
increased flux through transaminases will result in
stepped up ammonia output and increased availability of amino acid-derived carbons for oxidation or
anabolic pathways such as gluconeogenesis or glycogenesis. As indicated by the sea raven data (Vijayan
et al., 1996a), a certain percentage of the increased
nitrogen turnover may be retained as urea following
exposure to cortisol, with argininolysis, urea synthesis and breakdown of purines contributing to this
increase as judged from the increases in allantoicase
and arginase (Table 7).The physiological significance
of the production and retention of small amounts of
urea are not clear.
Functionally, some of the changes induced by
cortisol are surprising. In the case of tyrosine, for

Figure 3. Cortisol levels in plasma of rainbow trout (Oncorhynchus

mykiss) before and after 5 min of exhaustive exercise. The exercise
period is indicated by the vertical bar. Three groups of fish were
used. Groups were injected with saline (1 h before the exericse
period, ), metyrapone (2-methyl-1, 2-di-3-pyridyl-1-propanone),
an inhibitor of cortisol biosynthesis (1 h, ) or with dexamethasone
(24 h, 1). Vertical bars indicate SEM for six independent determinations. Cortisol concentrations in fish injected with dexamethasone
were all below the detection limit (5 ng mL1 ). Redrawn from
Milligan (1997).

instance, its carbon can only be used for oxidation or

ketogenesis, which teleost fish do poorly, and for gluconeogenesis, again not considered a major metabolic
route for species that generally do not control plasma
glucose nearly as tightly as mammals. Besides, tyrosine is not among the more abundant amino acids in
proteins in general. Similarly, in a study on tilapia hepatocytes in primary culture, we found that glutamine
synthetase could be induced by cortisol, but even after
induction, enzyme activities seemed much too low
to have any physiological importance (T.P. Mommsen
and M.M. Vijayan, unpublished data).
As mentioned above, recovery from exhaustive
exercise provides another good model for cortisol
dynamics, in that it includes, at least in the rainbow
trout, a transient tripling in endogenous cortisol concentration (Figure 3). As in the toadfish, this surge
in cortisol is correlated with changes in amino acid
metabolism, including increases in key amino acids in
plasma and in the liver. It should be noted, however,

245
that the pre-exercise levels of cortisol were already
several fold above resting concentrations reported for
this species, in fact higher than the reported postfeeding peak noted in other studies (Gamperl et al.,
1994). Thus, other important factors controlled by
cortisol may have been missed or were distorted
after the exhaustive exercise. The study by Milligan
is the first conclusive (with the proviso of an elevated baseline cortisol level) study for fish showing
production and efflux of glutamine together with
alanine from white muscle post-exercise. Unfortunately, cortisol involvement in this process could
not be identified unequivocally. While other evidence supports the idea of increased proteolysis, it
is not inconceivable that mass action governed by
large increases in muscle ammonia (Mommsen and
Hochachka, 1988) and pyruvate (Arthur et al., 1992)
without adjustments in enzyme titres of transaminases or glutamine synthetase, is responsible for
the increased glutamine and alanine output from the
muscle. Other cascading effects can be envisaged. For
instance, in the recovery state, cortisol may activate the proteolytic system responsible for activation
of AMP deaminase (Raffin and Leray, 1980), the
enzyme degrading AMP into IMP and ammonia, and
thus indirectly influence muscle pH (Mommsen and
Hochachka, 1988) and glutamine production. Regardless, the relative speed with which these postexercise
alterations come about seem to point to a nongenomic
action of cortisol. However, as cortisol is not the only
hormone changing during recovery (e.g. insulin and
epinephrine), it is quite possible that what is seen are
the results of interactions between cortisol and other
hormones.
The fact that both alanine and glutamine appear in
the plasma compartment after increases in endogenous
cortisol as part of the exercise response points to white
skeletal muscle as the most likely source and supports
the notion of peripheral proteolysis. Although Milligan and Girard (1993) did not detect any significant
decreases in muscle protein, we reiterate the above
argument and would not expect noticeable changes
in muscle because of its bulk, yet a significant effect
should be picked up in plasma. While the relatively
fast kinetics of the amino acid peak in plasma point
to liver with its high rate of protein turnover (Houlihan et al., 1995) as the likely source, we would like
to argue otherwise. As mentioned above, the liver
makes up only a small fraction of the fishs body by
weight (less than 2% in case of the rainbow trout), yet
after exercise plasma amino acid concentrations are

tripled, including those of L-alanine from 0.5 mM to

1.5 mM, notwithstanding unknown increases in flux.
Altogether, assuming a blood plasma volume of 5%,
substantial amounts of amino acid are mobilized. If
derived from liver, changes in liver protein should be
detected with ease. Yet total liver mass was unaltered
by implantation of cortisol into coho salmon parr or
smolts (Sheridan, 1986). However, if these amino
acids are mobilized from muscle, this amounts to an
insignificant amount of the protein present, because
muscle assumes about 20 times the bulk of the liver.
As often, the reality may lie somewhere between
the two extremes, with both liver and muscle contributing.
In developing embryos of the medaka (Oryzias
latipes, Advianichthyidae), (Cloud, 1981), exogenous
glucocorticoids induce precocious hatching, without
accelerating embryonic development. In addition to
these short-term actions, corticosteroids also exert
long-term effects on larval fish. In rainbow trout fry,
for instance, cortisol or cortisone exposure for 30
days leads to a significant masculinization noted over
half a year later, changing the sex ratio from 1:1 to
3:1 (cortisone) or 4:1 (cortisol). At the same time,
ovarian growth in the remaining immature females
was depressed significantly (Van den Hurk and Van
Oordt, 1985). Obviously, cortisol is plurifunctional
during the early development of fish, a conclusion that
is in line with evidence gathered for mammals. Studies on knockout mice with corticotrophin-releasing
hormone deficiency have pointed to the possibility
that corticosteroids exert their most important functions during fetal periods, rather than in postnatal life
(Muglia et al., 1995).
Our overall interpretation of the role of cortisol
of fish metabolism is summarized in Figure 4. This
figure attempts to reconcile the observations on peripheral proteolysis with widespread protein and amino
acid biosynthesis in liver, and to integrate changes in
enzyme activity with changes in carbon flux through
different pathways. Unfortunately, we feel we have to
shy away from a distinction between the housekeeping actions of this hormone and those apparent under
pharmacological conditions; there is simply not sufficient experimental evidence and at this point lines
of distinction would be quite arbitrary. Suffice it to
say that even small surges in cortisol are sufficient
to alter enzyme expression and metabolic output, and
that often such surges may form an inadvertent part of
experimental manipulation, thus occluding potential
finely tuned effects of the hormone.

246

Figure 4. Metabolic effects of cortisol in fish. The arrows indicate those pathways or processes that are either regulated or are potential sites (?) of action of cortisol. Dashed lines indicate flow or transport of metabolites. Abbreviations: FFA, free fatty acids; HOAD,
3-hydroxyacyl-coenzyme A dehydrogenase; GDH, glutamate dehydrogenase; GK, glycerol kinase; GNSase, glutamine synthetase; GPase,
glycogen phosphorylase; GSase, glycogen synthase; G6Pase, glucose 6-phosphatase; G6PDH, glucose 6-phosphate dehydrogenase; PEP
phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase.

247
Metyrapone, RU 486 and dexamethasone
In view of the severe limitations of the pharmacological approach to elucidate cortisols specific roles in
fishes, the alternative strategy would be to artificially
decrease plasma cortisol concentrations. One possible
route is to impose moderate exercise, which, among
other things, alters intraspecies interactions and tends
to increase growth performance (Christiansen and Jobling, 1990). After an initial, short-term overshoot
in plasma cortisol, species such as trout or salmon
respond to a long-term exercise regime with substantial decreases in plasma cortisol below control levels
(Woodward and Smith, 1985; Nielsen et al., 1994).
In the Atlantic salmon, cortisol levels were about half
those found in unexercised control fish (Boesgaard et
al., 1993). Unfortunately, some other common tools
used to define functions for other hormones are not
befitting for cortisol. For instance, it is impossible to
completely remove the cortisol-producing cells surgically owing to the diffuse nature of the interrenals in
teleosts, although the procedure has been tried in eels
(Butler et al., 1969). As far as we are aware, one of
the alternatives, immunoneutralization of cortisol, has
not been attempted for the fishes. Although, as noted
above, hypophysectomy will affect cortisol availability via different routes, cortisol is just one of a
multitude of hormones altered as a consequence of this
surgical intervention and it is not possible to establish
clear lines of evidence to explain observed metabolic changes. Therefore, the experimenters arsenal
is reduced to the use of inhibitors such as metyrapone, RU486 and analogues such as dexamethasone,
all of which have their own limitations (Gamperl et
al., 1994).
Metyrapone
Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone)
inhibits the 11- hydroxylase (Figure 1) together with
other cytochrome P450-dependent mono-oxygenases,
preventing the de novo synthesis of cortisol from
11-deoxycortisol. The compound has been used successfully to block cortisol synthesis in fishes, usually in short-term experiments, where it effectively
nullified the exercise-dependent peak in endogenous cortisol in trout (Figure 3) and the stress-related
peak in toadfish (Hopkins et al., 1995). The potential of metyrapone to lead to depletion of endogenous cortisol, owing to ongoing cortisol degradation
and inhibition of de novo synthesis, has yet to be
explored experimentally. But the in vivo studies by

Milligan and co-workers already hint this is a feasible, although a relatively lengthy, process. These
authors found only a slow decrease in plasma cortisol
within 8 hr of metyrapone application (Pagnotta et al.,
1994; Milligan, 1997). Despite its obvious usefulness,
some disconcerting interactions between metryapone
and glucocorticoid have been reported for mammalian
systems. Metyrapone functions as an inhibitor of cytochrome P450-dependent drug metabolism in mammalian microsomes (Netter, 1980), while inducing
the expression of CYP2B1, CYP1A1/2 and CYP3A,
an effect enhanced in the presence of hydrocortisone
(Wright et al., 1994). Metyrapone alone had no
effect on the expression of TAT, but in the presence of dexamethasone, it potentiated the induction
caused by the corticosteroids (Tongiani et al., 1998),
possibly by derepression of a GR-modulated factor
(Harvey et al., 1998). Finally, metyrapone alters
peripheral glucocorticoid metabolism itself by inhibiting 11-hydroxysteroid dehydrogenase (SampathKumar et al., 1996). Therefore, extreme care seems
advised when interpreting data obtained in experiments employing metyrapone and it cannot be tacitly
assumed that the observed alterations in metabolic
parameters are due solely to the inhibition of cortisol
biosynthesis.
Etomidate is another known inhibitor of adrenal
steroidogenesis in mammals. Although the compound
is widely used as a powerful anaesthetic for fish
(Akner et al., 1995), etomidate has not been used to
alter cortisol biosynthesis in interrenal cells.
An additional method to suppress endogenous
cortisol is by the use of corticostatin a potential
member of the defensin family of peptides. Mammalian corticostatins bind selectively to the ACTH
receptor, inhibiting ACTH-mediated synthesis of steroids (Solomon, 1993). The corticostatin-related peptide isolated from the sea lamprey contains the key
basic residues mediating the antisteroidogenic action
of mammalian corticostatins (Segner and Braunbeck,
1988), making this a promising tool to selectively
manipulate endogenous cortisol concentrations.
RU 486
RU 486 (RU 38486, Mifepristone) is an antiglucocorticosteroid that exerts its effects at the receptor
and possibly the postreceptor level of steroid action.
The molecule has a phenyl-amino-dimethyl group at
the 11-position of the steroid backbone and this,
plus other substitution, is critical to the antiglucocorticosteroid effects of RU 486. Its major application,

248
however, is as an antiprogesterone in contraception,
with its full clinical potential having been held back
by its manufacturer (Baulieu, 1997). RU 486 blocks
tissue GRs, while also impeding the negative feedback
effects of cortisol on the hypothalamic-pituitary axis,
resulting in increases in ACTH release and enhanced
plasma cortisol values, at least in mammals (Baulieu,
1997).
This antihormone has been used in a number of
fish studies to block the effects of exogenously added
cortisol and to confirm conclusively that cortisol was
indeed the active principle behind the noted effects.
In competitive binding assays RU 486 displaces
3 H-cortisol (Pottinger, 1990) and 3 H-dexamethasone
(Lee et al., 1992) from rainbow trout liver cytosol
and 3 H-TA from whole-brain cytosol of chinook salmon (Knoebl et al., 1996). In most instances, its
binding properties are similar to those of the native ligand and therefore RU 486 was instrumental to
confirm that cortisol binding was a type II or GRlike rather than a type I or mineralcortocoid receptor.
Interestingly, Knoebl et al. (1996) also used RU
28362, an apparent pure synthetic glucocorticoid
that is as effective as corticosterone; this compound
was 5 times less effective at displacing 3 H-TA than
was RU 486. Dasmahapatra and Lee (1993) reported that RU 486, but not spironolactone, a type I (or
MR) glucocorticoid receptor antagonist, blocked the
dexamethasone-induced decreased in trout hepatocyte
CYP1A1 protein and 7-ethoxyresorufin-O-deethylase
(EROD) activities. Administration of RU 486 using
slow-release oil implants also prevented the cortisolinduced hyperglycaemia observed in fasted rainbow
trout (Reddy et al., 1995). Clearly, RU 486 provides
an excellent experimental tool to block GR-induced
activities in the fish system.
The antagonistic effects of RU 486 do not prevail
in all cases. RU 486 does exert some glucocorticoidlike effects by increasing hepatosomatic index, hepatic
glycogen and glyceraldehyde-3-phosphate dehydrogenase activities in brook charr (Vijayan and Leatherland, 1992). Similar glucocorticoid actions of RU 486
were also noted for carbohydrate metabolism in isolated trout hepatocytes (Vijayan et al., 1994b). It
appears that RU 486 can block some effects of cortisol,
but does not share some glucocorticoid-like effects
with the natural agonist. Exactly how or why RU 486
might have this dual role is unclear. In live mammalian cells containing a green fluorescent protein
fused to the rat GR, RU 486 bound to this complex
and was translocated into the nucleus (Htun et al.,

1996). However, unlike the agonist dexamethasoneGR complex that showed nuclear foci and presumed
DNA binding, the RU 486 complex appeared to bind
to the nuclear matrix in a diffuse manner. Therefore it
seems that agonist and antagonist binding to the GR
are quite distinct, at least in mammals (Moudgil and
Gunda, 1991; Htun et al., 1996). Whether a similar
situation occurs in fish is not known, as it may be
that the fish GR is simply more promiscuous, resulting in RU 486-activated transcription. The utility of
RU 486 in cortisol physiology must therefore be considered with regard to this apparent dual role of the
antagonist.
Dexamethasone
Finally, there is the two-faced synthetic glucocorticoid
dexamethasone. This compound is effective to depress
endogenous cortisol release by blocking the pituitaryinterrenal axis (Pickering et al., 1987), primarily by
inhibiting the release of ACTH. However, it also acts
like a glucocorticoid in its own right, as is obvious
from the many cortisol-like effects of dexamethasone
listed in Table 5 to 8, and its ability to bind selectively
to the fish glucocorticoid receptor (Table 3). Therefore, its application to prevent cortisol release from
the interrenals may simply replace the natural hormone with a synthetic version. Nevertheless, some
differences in their relative effectiveness are apparent
in fishes, especially at the GR level (Table 3). In the
exhausted rainbow trout, for instance, dexamethasone
does not substitute for the proteolytic action of the
natural compound (Milligan, 1997). Dexamethasone
binds to GR as effectively as cortisol, and in most
cases even more tightly (Table 3). As a result, it is
not unreasonable to expect cortisol-like actions of the
analogue even if endogenous plasma cortisol is nonexistent. From the total removal of the natural hormone from the plasma compartment, it can be demonstrated that in the course of the 24 h pretreatment
period, dexamethasone has replaced cortisol on its
receptors or the receptors are eventually unoccupied.
Unfortunately, the turnover rate of dexamethasone
itself in fishes remains to be determined.
Some of the apparent differences noted between
the alleged actions of cortisol and those of dexamethasone may be explained by different uptake kinetics for the two compounds, at least in trout liver. In
one of the first studies on isolated trout hepatocytes,
cortisol uptake was linear for only 10 min, albeit much
slower than in rat hepatocytes, and then fell off dramatically over the following few minutes. In contrast,

249
dexamethasone uptake was somewhat faster and continued at only slightly reduced rates for at least 30 min
(Porth-Nibelle and Lahlou, 1981). Hence, it can be
assumed that the synthetic glucocorticoid can accumulate to much higher concentrations than the natural
compound.
Simply from the fact that a given compound will
bind to the GR, as triamcinolone (TA) does very
effectively (Table 3), one cannot deduce automatically that the compound can replace a natural hormone
in its various functions equally well. A comparison
between the synthetic and natural ligands of the GR
is complicated by the fact that dexamethasone and
triamcinolone both show high affinity for corticosteroid receptors, yet display relatively low affinity for
plasma steroid-binding protein. In rat hepatocytes,
TA, dexamethasone and cortisol will bind to the GR
very effectively, but orders of magnitude differences
exist in their relative potency to induce TAT (TA
0.8 nmol L1 ; dexamethasone 3.3 nmol L1 ;
cortisol 693 nmol L1 ). As well, the ability to
induce cytochrome P450 synthesis in rat hepatocytes
varies substantially between the three compounds
(TA 9.5 mol L1 ; dexamethasone 0.7 mol
L1 , cortisol 50 mol L1 ; Schuetz and Guzelian,
1984). Instead of being constant, the resulting ratios
(P450/TAT) for these three model compounds are TA
12 000, dexamethasone 212, and cortisol 72. In
addition, the induction of TAT by dexamethasone in
mammals is direct, while the induction of PEPCK by
dexamethasone is noticed only in the presence of glucagon (Iynedjian et al., 1985). Again, we have no idea
whether similar factors are operating in the piscine
system.
Lipids
As with the other metabolic pathways in fish, the regulation of lipid metabolism by cortisol is not without
controversy, making it difficult to develop any congruent picture. Again, it is not clear whether experimental
or species differences are at the root of these contradictory results. The prevailing notion assigns a strong
peripheral and hepatic lipolytic action to cortisol, resulting in increases in plasma un-esterified fatty acids.
The fish liver seems to be the main target, coping with
the increased availability of fatty acids through oxidation and possibly resynthesis, while the energetically
less important glycerol provides an ideal gluconeogenic substrate. Some indirect and selected direct
evidence supports this metabolic scenario. In cortisol-

fed channel catfish, the liposomatic index drops by

at least 50% (Davis et al., 1985), and European eels
and salmon parr show increased peripheral lipolysis in
the presence of elevated cortisol (Dave et al., 1979;
Lidman et al., 1979; Sheridan, 1986), at times correlated with decreasing plasma triacylglycerol levels
(Lidman et al., 1979). Cortisol injections also activate triacyglycerol lipase in mesenteric fat, red muscle
and liver of coho salmon parr, and subsequently, liver
triacylglycerols and phospholipids decrease, whereas
in the muscle, only the triacylglycerol levels but
not the phospholipids are negatively affected by the
steroid (Sheridan, 1986). Not surprisingly, none of
these effect is noted in coho salmon smolts with
their naturally elevated cortisol levels (Shrimpton et
al., 1994). These smolts are already in a state of
cortisol-dependent activation, reflected in lower levels
of hepatic lipid and red muscle triacylglycerols and
increased activity of triacylglycerol lipase (Sheridan,
1986). The results for the salmon parr confirm previous observations on cortisol-dependent increases in
titres of plasma fatty acids in goldfish (Minick and
Chavin, 1969) and some eels (Butler, 1973; Dave et
al., 1979; Lidman et al., 1979), but not all (Larsson
and Fnge, 1977).
The fate of the increased fatty acids appears to
vary between species, with oxidation, re-esterification
and general turnover competing for the substrates. For
instance, hydroxyacyl coenzyme A dehydrogenase
(HOAD), a key mitochondrial enzyme of fatty acid
oxidation, is increased in the presence of corticocsteroid in sea raven and charr (Table 7), but not
in two closely related tilapia (M.M. Vijayan and A.
Takemura, unpublished data; M.M. Vijayan and T.P.
Mommsen, unpublished data) or toadfish (Mommsen
et al., 1992). Additionally, enzymes involved in hepatic fatty acid biosynthesis may be induced. One
example is glucose 6-phosphate dehydrogenase, a
cytosolic enzyme contributing reducing power to lipid
synthesis (Table 7) and whose activity is elevated
in a number of fish species, but not in nile tilapia
(Oreochromis niloticus, Cichlidae) (M.M. Vijayan and
T.P. Mommsen, unpublished data). Another is malic
enzyme, also potentially involved in redox balance.
Its activity is increased in cultured tilapia hepatocytes. Furthermore, re-esterification of the liberated
and newly synthesized fatty acids may be favoured
through the observed cortisol-dependent increases in
glycerol kinase (Vijayan et al., 1991). The end result would be a build-up of hepatic lipids under the
influence of cortisol, a fact that indeed has been

250
confirmed for three species of anguillid eels Japanese (Chan and Woo, 1978), European (Olivereau,
1966) and American (Butler, 1973; Foster and Moon,
1986). Contrasting to this picture is the apparent failure of postspawn, hypercortisolaemic pink salmon
(Oncorhynchus gorbusha, Salmonidae) to synthesize
triacyglycerols (Phleger, 1971) and the absence of
cortisol-dependent changes in liver lipid in rainbow
trout (Netter, 1980). This latter observation concurs
with the situation in mammals, where cortisol, perhaps
predominantly, interferes with the re-esterification of
free fatty acids, leading to the same end result, namely
elevated titres of plasma fatty acids.
Stepped-up turnover of triacylglycerols and phospholipids will also deliver glycerol. However, because
cortisol appears to increase the titres of hepatocyte glycerol kinase and -glycerophosphate dehydrogenase in some species (Vijayan et al., 1991), predictions about the overall effect on the static concentration of plasma glycerol vary. These predictions will
range from increases owing to induction of delivery
pathways (i.e. lipase) to decreases owing to increased
turnover (i.e. lipase plus glycerol kinase, etc.) as noted
for the American eel (Foster and Moon, 1986). Once
phosphorylated, the glycerol can be used for gluconeogenesis or for the biosynthesis of triacylglycerols; as
shown above, both pathways are affected profoundly
by the hormone.
A useful tool to identify specific functions of
cortisol is hypophysectomy, which eliminates crucial
pituitary hormones and with it ACTH, thus leading
to reduced levels of plasma cortisol (Nishioka et al.,
1985). This reduction, in turn, results in osmoregulatory failure of coho salmon smolts in SW and dramatically depresses liver triacylglycerol lipase. Interestingly, cortisol replacement therapy can nearly restore
the triacylglycerol lipase activity to normal levels
for smolts (Sheridan, 1986), likely providing a unique
glimpse into one of the housekeeping functions of
cortisol.
The fact that oxygen consumption increases with
cortisol exposure has been mentioned elsewhere in
this review, but in the context of lipid metabolism
it is interesting to note that the respiratory quotient is also affected. In intact or hypophysectomized
Japanese eels, the normal respiratory quotient (RQ)
drops from 0.80.85 to below 0.65 (Chan and Woo,
1978), strongly implying that at least a portion of the
increased oxygen consumption is met by the oxidation of fatty acids. One wonders which pathways are
involved in this increased oxygen consumption: likely

candidates are gluconeogenesis and protein synthesis

in the liver, protein synthesis in the gill and fatty acid
synthesis in the liver.
Mammalian models show some similar, but also
additional, actions for corticosteroids on lipids. For
instance, the administration of cortisol or dexamethasone profoundly alters the dynamics of lipoprotein lipase (LPL) in human adipose tissue, especially in the presence of insulin (Ottosson et al.,
1994). In addition to increasing the enzyme activity, the hormone also enhances the amount of tissue
LPL mRNA and the relative rate of LPL synthesis.
These overall effects seem to be achieved largely
through inhibition of LPL degradation. In conjunction with each other, glucocorticoids and insulin act
as causative agents in the differentiation of human
adipocytes from precursor cells (Hauner et al., 1987).
In cultured rat hepatocytes, glucocorticoids potentiate the ability of insulin to stimulate lipogenesis
by activating a number of enzymes, including glucose 6-phosphate dehydrogenase (Stumpo and Kletzien, 1984), pyruvate kinase (Fleig et al., 1984),
malic enzyme, -glycerophosphate dehydrogenase
(Wilson and McMurry, 1981) and acetyl coenzyme A
carboxylase (Vernon et al., 1991). In fetal rat lung and
hepatocytes, an additional target is fatty acid synthase
(Roncero et al., 1989; Xu et al., 1993).

Interactions with other hormones

Glucocorticoids are known to have permissive effects
with regards to other metabolic hormones. Although
similar interactive effects have been demonstrated in
some fish hepatocyte studies, the precise mechanisms
responsible for these interactions in fish are unknown
(Mommsen et al., 1992; Vijayan et al., 1993b, 1994b).
This, however, is not the case for mammals.
Glucocortocoids and insulin are major antagonistic
regulators of energy metabolism in mammals, probably acting in the brain, possibly through neuropeptide
Y (NPY) (Strack et al., 1995). Many studies have
explored the basis for this reciprocity, primarily using
the two enzyme models, PEPCK and TAT, that are
oppositely affected by the two hormones. Glucocorticoids increase while insulin decreases both PEPCK
(Sutherland et al., 1996) and TAT (Ganss et al., 1994)
gene transcription and glucocorticoid activation of
these enzymes in liver. A major problem in these
studies is that increased plasma glucocorticoid leads
to hyperinsulaemia, but recent studies provide evid-

251
ence that the pathways for the effects of the two
hormones are quite distinct. For example, rat hepatoma cells exposed to dexamethasone showed significant increases in IRS-1 protein and phosphorylation levels, and greater association between IRS-1
and phosphatidylinositol 3-kinase (Saad et al., 1995),
but prolonged insulin exposure did just the opposite.
If insulin and dexamethasone were added together,
insulin effects always predominated. This would
indicate that the major interaction between these two
hormones is at the level of their respective signalling
systems. Yet, other evidence shows a GRE located
on the insulin receptor gene (Lee and Tsai, 1994)
and a potential interplay between a glucocorticoidresponsive element and the cAMP-responsive element
(CRE) (Ganss et al., 1994), suggesting that interactions between transcription-binding sites on sensitive
genes are also important to the insulin-glucocorticoid
antagonism. Certainly, direct competition between a
GRE and a CRE is a simple yet elegant mechanism to
account for this antagonism (Villafuerte et al., 1995).
As mentioned elsewhere, in mammals as in fishes,
exogenous cortisol or elevated endogenous cortisol
will be accompanied by hyperglycaemia. In mammalian models, this hyperglycaemia, in turn, is partially responsible for the concurrently observed hyperinsulinaemia. However, the increased plasma concentrations of insulin (and C-peptide) fail to effectively
counteract the hyperglycaemia, a metabolic situation
for glucose metabolism known as glucose resistance
and resulting in so-called cortisol-diabetes (Saad et al.,
1995). Apart from the hyperglycaemia accompanying
hypercortisolaemia, all other parameters involved in
the mammalian scenario show striking differences in
the piscine situation. First, glucose is not as powerful an insulinotrophin as in the mammals and hence
smaller, if any, hyperinsulinaemia can be expected
via this route; second, teleost fishes, with their generally high (by mammalian standards, excessive) plasma
insulin concentrations and their slow response to exogenous insulin, can be considered insulin resistant
even in the absence of exogenous glucocorticoids
(Mommsen and Plisetskaya, 1991). Both these differences make it unlikely that insulin will have any
counterregulatory effects on cortisol-induced hyperglycaemia. Having said this, however, the concurrent
cortisol-dependent proteolysis may somewhat compensate for the relatively poor insulinotrophic action,
because key amino acids, especially arginine and
lysine, are powerful insulinotrophins in the fishes
(Ince and Thorpe, 1977; Ronner and Scarpa, 1987);

unfortunately, the overall metabolic outcome seems

to be unpredictable. Because of this relative insulin
resistance, hyperinsulinaemia accompanying hypercortisolaemia in fishes may influence amino acid
turnover and some growth-related functions. Glucocorticoids may directly oppose insulin actions as
shown for sheep adipocytes, where dexamethasone
prevents the time-dependent decrease in insulin binding (Wastie et al., 1995). In contrast, dexamethasone
has been shown to increase insulin binding to a
number of different cell types; it does this largely
by increasing the rate of insulin receptor transcription, but also by preventing receptor degradation. In
recent years, the potential interactions between insulin
and glucocorticoids, and incidentally also androgens,
have received another twist. A previously discovered
receptor accessory factor (RAF), which enhances the
binding of glucocorticoid receptor fragments to DNA,
was found to be virtually identical to insulin-degrading
enzyme and to possess the ability to split insulin proteolytically. Moreover, insulin was found to compete
with the RAF and GR interaction (Kupfer et al., 1994).
Although no direct metabolic effects of dexamethasone were noted in the toadfish, this synthetic
corticosteroid seems to prevent some of the changes
brought about by glucagon, specifically glucagondependent alterations in the activities of key enzymes
normally associated with lipid metabolism (Mommsen
et al., 1992). A few observations were surprising.
For instance, dexamethasone exerted no effects no
PEPCK, even in the presence of glucagon; in mammals, the corticosteroid potentiates the glucagondependent induction of the enzyme. Further, the activities of hepatic glucose 6-phosphate dehydrogenase
and malic enzyme, two enzymes feeding reducing
equivalents into the cytosolic pathways and thus useful
indicator enzymes for fatty acid biosynthesis, showed
opposing trends: activity of G6PDH decreased significantly, while malic enzyme increased (Mommsen
et al., 1992). Because glucagon and glucagon-like
peptide-1 in fishes affect largely common metabolic
targets (Plisetskaya and Mommsen, 1996), it would
be interesting to analyse the extent to which cortisol,
glucagon and GLP-1 interact, and whether similar
negative feedback on the expression of glucagon-like
peptide-1 receptor by corticosteroid exists as reported
for a mammalian cell line (Richter et al., 1989).
Glucocorticoids affect the mitogenic actions and
bioavailability of insulin-like growth factor 1 (IGF-1).
Dexamethasone decreases IGF-1 production in osteoblasts at the level of transcription, probably by binding

252
to a GRE located on the IGF-1 gene (Delany and
Canalis, 1995). At the same time, dexamethasone
increases IGF-1 receptor phosphorylation and signalling in muscle cells (Georgino and Smith, 1995).
One of the largest effects appears to be the decrease
in levels of IGFBP-3, the principal IGF-binding protein found in mammalian plasma. This effect occurs
at the level of IGFBP-3 gene transcription in hepatoma cells, resulting in an increase in circulating and
thus bioactive IGF-1 in the plasma (Villafuerte et al.,
1995). As yet, the precise mechanism to account for
this decrease is unknown. The area of IGF-binding
proteins is actively pursued in fishes (Duan, 1998) and
there is evidence for an IGFBP-3-like protein in some
species (Siharath and Bern, 1993).
Glucocorticoids also interact with other hormones
at various points within their mechanisms of action.
Growth hormone receptor numbers are decreased by
glucocorticoids (King and Carter-Su, 1995) and a
GRE has been localized to the first intron of the
human growth hormone gene (Burnstein and Cidlowski, 1989). Somatostatin mRNA levels are reduced
by glucocorticoids, possibly by a direct interaction
between GRE and CRE binding sites. Finally, glucocorticoids accelerate the maturational change between
- and -adrenoceptors in neonatal rat liver (Huff et
al., 1991).
This multitude of interactions between glucocorticoids and other metabolic hormones in mammals
is obviously critical to development and to overall
energy balance. Whether this is the case in fish is
totally unknown. As demonstrated below, cortisol
modulates the actions of other hormones on cell metabolism, growth and development, and stress (including toxicants) in fishes, but often these effects were
analysed in isolation, and even more often, mechanistic analyses were beyond the scope of the piscine studies. Whether, as in mammals, the observed
effects represent direct interactions between cortisol,
the GR or GRE and these hormones, needs to be identified. Dexamethasone was found to down-regulate
cytochrome P4501A1 protein and EROD activities in
cultured trout hepatocytes (Dasmahapatra and Lee,
1993). The authors proposed that, as with mammals,
the CYP1A1 gene in trout contains a GRE for GR
binding; direct evidence for this is lacking at the
present time. As noted above, a GRE-like motif was
found on the rainbow trout prolactin gene (tPRL) promoter (Argenton et al., 1996) and it is elements like
these that need to be identified before an understanding of the mechanism of action of glucocorticoids

in fish is possible. Obviously, functional GREs hold

the key to how cortisol and other glucocorticoids
demonstrate such permissive effects.

Physiological role of cortisol

Glucose regulation during stress
Glucocorticoids are vital for maintaining basal and
stress-related homeostasis in mammals. Under resting conditions, cortisol sustains normoglycaemia and
prevents arterial hypotension. In the stressed state,
elevated cortisol is important for central nervous system activation, increasing blood glucose concentration
and elevating mean blood pressure, all of which are
important for coping with stress (Bamberger et al.,
1996). Cortisol is also thought to curtail the stressinduced inflammatory/immune reaction that might
otherwise lead to tissue damage (Bamberger et al.,
1996). In fish, the role of cortisol in increasing plasma
glucose concentration is becoming apparent and the
mechanisms involved are being elucidated. Stress is an
energy-demanding process as studies have shown that
stress increases metabolic rate and oxygen uptake in
fish (Barton et al., 1987). To cope with the increased
energy demand, fish mobilize substrates to fuel cellular processes. Glucose is an important fuel for
metabolism and certain tissues may rely primarily on
glucose (for example brain, heart, blood cells, and
gills; Mommsen, 1986). Unfortunately, in fish the
peripheral utilization of glucose is not clearly understood, especially during stress, and is one research
area that needs to be studied in greater detail, although
glucose is likely overrated in its importance to piscine
metabolism in general. With the exception of the metabolically unusual American eel (Cornish and Moon,
1985) and skipjack tuna (Katsuwonus pelamis, Scombridae) (Weber et al., 1986), glucose turnover rates
are one to two orders of magnitude lower than those
for resting mammals of comparable size, and elevated
plasma cortisol levels failed to alter glucose turnover
in sea raven (Vijayan and Moon, 1994) or rainbow
trout (Andersen et al., 1991). Although glucose utilization for individual tissues can increase dramatically,
as in red muscle during exercise, West et al. (1993)
report that even at the high rate glucose accounts for
less than 10% of the oxidative metabolism of this tissue, although values as high as 2040% have also been
reported for this same species (Lauff and Wood, 1996;
Kieffer et al., 1998).

253
Based on the role of cortisol on intermediary
metabolism, we suggest a working model to elucidate the hyperglycaemia role of cortisol during stress
in fish (Figure 4). Elevated cortisol during stress
may play a role in the immediate production of
glucose by increasing glycogenolysis. This effect of
cortisol is perhaps nongenomic, involving alteration
in the phosphorylation-dephosphorylation status of
glycogen phosphorylase as shown in rat hepatocytes
(Gomez-Munoz et al., 1989). In addition, cortisol
may have a permissive effect on epinephrine and/or
glucagon-mediated glycogenolysis (Reid et al., 1992;
Vijayan et al., 1993b). The mechanism involved may
be an increase in the recruitment/redistribution of
surface receptors for these catabolic hormones with
cortisol, thereby increasing the responsiveness of the
cells (Reid et al., 1992). However, it is also possible that there are postreceptor alterations sensitizing
the cells to the actions of epinephrine and glucagon, but such studies are yet to be carried out in
fish cells. The immediate increase in plasma glucose
concentration as a result of glycogenolysis would suggest a decrease in liver glycogen content post-stress,
although that does not appear to be the case in fish
(Vijayan et al., 1994a). We hypothesize that the repletion/maintenance of glycogen, as well as the maintenance of plasma glucose concentration after stress, is
mainly attributed to cortisol, perhaps in conjunction
with insulin. Recent studies for the first time show
clearly a very potent interaction between cortisol and
insulin on glycogen deposition in primary cultures of
tilapia hepatocytes (M.M. Vijayan and A. Takemura,
unpublished data). Unfortunately the impact of stress
or elevated cortisol levels on plasma insulin concentration in fish is unknown. In mammals, glucocorticoid
treatment does result in hyperinsulinaemia.
Vijayan et al. (1994a) showed that glucose production was no longer responsive to epinephrine and
glucagon 3 h after a handling disturbance, whereas
production in response to insulin was significantly
increased. This increased responsiveness to insulin
after stress may prevent glycogen breakdown and promote glycogen synthesis (Pereira et al., 1995), resulting in net glycogen conservation. As cortisol increases
the gluconeogenic capacity of the cells, some of these
C3 precursors are perhaps channelled for glycogen
repletion in addition to increasing glucose production. In support of this hypothesis, there was increased
gluconeogenesis from alanine in hepatocytes at 3 h
after the handling disturbance. It is not known if this
increased gluconeogenesis is due to cortisol-induced

gene transcription; in a companion study, RU 486

significantly blocked the cortisol-induced gluconeogenesis from alanine in trout hepatocytes, arguing for
a steroid-receptor-mediated process (Vijayan et al.,
1994b).
In addition to increasing gluconeogenic capacity,
cortisol has also been shown to increase amino acid
mobilization/catabolism in fish. The source of this
amino acid pool has not been clearly demonstrated
but it is conceivable that free amino acids originate
from the muscle pool, as mammalian studies have
clearly shown increased peripheral proteolysis associated with hypercortisolaemia (Brillon et al., 1995).
Cortisol may play a dual role by increasing the gluconeogenic capacity of the liver and by providing
gluconeogenic precursors from peripheral stores as
suggested in mammals (Fujiwara et al., 1996). There
is, however, a dearth of information as to the role
of cortisol on peripheral substrate mobilization and
the hepatic utilization of these substrates for gluconeogenesis and oxidation in fish. Lactate may also be
a key substrate for gluconeogenesis. After exercise,
cortisol increases muscle glycolysis and plasma lactate concentration in rainbow trout (Eros and Milligan,
1996). Lactate is a preferred substrate for gluconeogenesis in fish (Suarez and Mommsen, 1987), and
lactate gluconeogenesis is stimulated by glucocorticoid (Renaud and Moon, 1980; Janssens and Waterman, 1988; Mommsen et al., 1992). Consequently,
it is arguable whether lactate is utilized for glucose
production/liver glycogen conservation after stress in
fish. The role of lipids in the cortisol-induced gluconeogenic process is not clear, but two independent lines
of evidence point towards glycerol as an important
player. Cortisol-treated brook charr showed increased
activities of glycerol kinase and glyceraldehyde-3phosphate dehydrogenase activity in the liver (Vijayan
et al., 1991); both enzymes are essential to funnel glycerol carbons into gluconeogenesis, although glycerol
kinase is also important in the route to re-esterification
of fatty acids. As shown above, cortisol increases the
rate of lipolysis in fish and the resulting glycerol could
be utilized for gluconeogenesis. However, further
study is necessary to understand the role of cortisol
on glycerol utilization in fish and the potential partitioning of glycerol between gluconeogenesis and lipid
synthesis.
Thus, one of the important metabolic roles of
cortisol during stress is in the glucose-regulation
and glycogen-repletion processes, both of which are
important pathways for the recovery from stress. This

254
action of cortisol probably is in conjunction with other
glucoregulatory hormones such as epinephrine, glucagon and insulin; the control mechanism(s) might
include alterations in hepatocyte responsiveness to
these hormones. Cortisol may also be playing a role
in the peripheral mobilization of substrates, thereby
providing precursors for hepatic gluconeogenesis in
fish.
Toxicant exposure
The metabolic role of cortisol described above could
have a profound effect on the recovery of fish from
subsequent stressors. Studies indicate that toxicants
may have a significant effect on cortisol dynamics
in fish. Two species of perch, sampled from sites
polluted by high levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls and mercury, failed to show a cortisol response to acute
stress compared with fish from reference sites (Hontela et al., 1992). The toxicant-exposed fish also had
atrophied pituitary corticotropes. It appears that prolonged hyperactivity of the cortisol-producing cells
associated with long-term pollutant exposure will lead
to an exhaustion of the pituitary-interrenal axis both
in mammals (Bamberger et al., 1996) and in fish
(Hontela, 1997). Recently we exposed fish to TCBP
and examined the potential of trout hepatocytes for
cortisol uptake and metabolism (Vijayan et al., 1997b).
The results showed higher uptake and metabolism in
TCBP-exposed fish, which might result in cortisol
being unavailable for normal physiological actions.
To address the possibility of this change in hepatocyte potential being associated with cytochrome P450
induction, we treated trout with -NF (a potent P450
inducer) and examined cortisol dynamics (Wilson et
al., 1998). -NF exposure in vivo abolished interrenal
sensitivity to ACTH stimulation in vitro. Furthermore, -NF-exposed fish showed a muted cortisol
response to a handling stress compared with the shamtreated fish. These results indicate that contaminants
may affect: (1) the pituitaryinterrenal axis in fish,
resulting in lower cortisol production; and (2) the
hepatocyte potential for cortisol clearance in fish,
resulting in attenuated cortisol action. These studies do not clearly indicate if the changes seen were
dependent upon activation of the cytochrome P450
system.
The enzymes encoded by the cytochrome P450
family of genes (CYP) play a very important role
in the metabolism of lipophilic xenobiotics, includ-

ing PAHs (Stegeman, 1993; Buhler and Wang-Buhler,

1998). The induction of CYP1A protein in fish tissues
is important for the metabolism and biotransformation of xenobiotics in the aquatic environment. Recent
studies show that glucocorticoids potentiate the PAH
induction of CYP1A by a classical glucocorticoid
receptor mechanism in fish cell lines (Celander et al.,
1996). While dexamethasone by itself did not affect
CYP1A induction in these cell lines, Dasmahapatra
and Lee (1993) described a significant dose-related
reduction in CYP1A protein and EROD activity with
dexamethasone in trout hepatocytes. The potentiating effect of cortisol on -NF-induced activity of
EROD (a CYP1A-mediated activity) was observed
in primary cultures of trout hepatocytes (Devaux et
al., 1992). These results suggest an adaptive role for
elevated cortisol in the biotransformation of xenobiotics and the subsequent elimination of toxicants in
fish. The inhibition of the cortisol response in fish
exposed to polluted sites (Hontela et al., 1992) or to
-NF (Wilson et al., 1998) may, therefore, have a
detrimental effect on the biotransformation processes
for PAHs in fish. Certainly research is warranted to
understand the lack of cortisol response on PAH toxicity, metabolism and elimination in fish (Hontela,
1997).
Osmoregulation
Cortisol is considered a SW-adapting hormone as
cortisol treatment has been shown to increase SW tolerance in fish. Also, plasma levels of cortisol increase
during the parrsmolt transformation in salmonids and
the increase in plasma cortisol concentration correlates
with the onset of hypo-osmoregulatory abilities in SW.
The higher plasma cortisol concentration may be due
to the permissive effects of pituitary and extrapituitary
hormones on interrenal cortisol secretion in SW. One
of the mechanisms by which cortisol adapts fish to
SW is by cellular differentiation of chloride cells and
by stimulating the branchial Na+ /K+ -ATPase activity (reviewed by McCormick, 1995). A recent study
demonstrated that this cortisol-induced increase in
gill Na+ /K+ -ATPase activity is in part due to the
expression of -subunit mRNA of Na+ /K+ -ATPase
(Madsen et al., 1995). These actions of cortisol
are perhaps receptor mediated: studies have shown
increased cortisol receptor function in SW acclimated
fish gills (Weisbart et al., 1987) and developmentally during the parr-smolt transformation (Shrimpton
et al., 1995). A recent study also showed GR gene

255
expression in chum salmon chloride cells (Uchida
et al., 1998), supporting a direct effect of cortisol
on chloride cell function in fish (McCormick, 1995).
RU 486 inhibited the hypo-osmoregulatory effect of
cortisol in Atlantic salmon, clearly arguing for a
cortisol receptor-mediated effect on SW acclimation
in fish (Veillette et al., 1995). It has been established that the SW acclimation process is not solely
owing to cortisol, but involves interaction with other
hormones (McCormick, 1995). Recent studies demonstrate a synergy between cortisol and other hypoosmoregulatory hormones such as growth hormone
(GH) and insulin-like growth factor-1 (IGF-1) on SW
tolerance in fish. Cortisol had a synergistic effect with
GH on increasing gill Na+ /K+ -ATPase activity and
salinity tolerance in Atlantic salmon, while IGF-1
was less potent than GH (McCormick, 1996). Similar synergism between cortisol and GH on Na+ /K+ ATPase, chloride cell numbers and salinity tolerance has also been reported in brown trout (Madsen,
1990).
Cortisol receptor gene expression was noted in
the pavement cells of chum salmon fry (Uchida et
al., 1998). Pavement cells are the predominant site
for H+ -ATPase activity, although recent studies have
shown that these ATPases are immunolocalized in
both chloride and pavement cells in rainbow trout
(Lin et al., 1994; Sullivan et al., 1995). The presence of cortisol receptor gene expression in pavement cells (Uchida et al., 1998), coupled with the
observation that cortisol infusion increased gill H+ ATPase activity in FW trout (Lin and Randall, 1993),
argues for a role for cortisol in pavement cell function and sodium uptake in FW fishes. Cortisol has
been previously shown to stimulate whole-body calcium uptake and the branchial calcium pump in FW
rainbow trout (Flik and Perry, 1989). These results
suggest the possibility that cortisol is also an important hormone for FW acclimation in fish. It is not
known if this action is independent of prolactin stimulation, although elevated cortisol has been shown to
inhibit prolactin secretion from tilapia pituitary glands
(Borski et al., 1991).
Despite a large body of work on the effect of
cortisol on osmoregulation in fish, very little is
known about the metabolic role of cortisol during
ionoregulation in fish. Because activation of branchial
pumps requires energy, it is likely that the elevation of plasma cortisol observed in SW assists in
the energy re-partitioning process. Glucose appears
to be a preferred oxidative substrate for gill metabol-

ism (Mommsen, 1984; Perry and Walsh, 1989) and

cortisol may be involved in the production of glucose
to provide energy for protein synthesis and sodium
pump activation in SW. As a consequence, the nutritional state of the animal may be very important in
the SW acclimation process. A recent study suggests
the possibility that the energy demand during SW
transfer is higher in food-deprived fish, resulting in
an elevated cortisol response and cortisol-associated
metabolic effects to cope with the stress of SW transfer (Vijayan et al., 1996b). Certainly more research
is needed to elucidate the pathways that are triggered
by cortisol, especially the uptake of substrates into
the gills (transporters?), and the absence of a cortisol
response on these processes in SW fish.
Growth and reproduction
Glucocorticoids are known to suppress reproductive
functions, forming part of the mechanisms involved
in delaying reproduction at times of stress. This
phenomenon appears to be an adaptive response to
divert metabolic building blocks away from biosynthetic pathways. In mammals, such glucocorticoid
diversions have been noted for several tissues. Dexamethasone negatively affects the abundance of IGF-1
mRNA in neural cells (Adamo et al., 1988), and
in liver it inhibits the growth hormone-dependent
increases in IGF-1 mRNA and interferes with the transcription of the oestrogen receptor gene. In the uterus,
the compound attenuates IGF-1 transcription induced
by oestradiol (Sahlin, 1995).
Exogenous cortisol in fishes increases the hepatic
transcription of the vitellogenin gene, albeit without a
concomitant enhancement of liver or plasma vitellogenin in treated fishes (Ding et al., 1994). Not only
does this observation support the notion of nuclear
actions of cortisol, it also implies a hidden metabolic
cost owing to the futile synthesis of the vitellogenin
mRNA. At the same time, cortisol appears to suppress plasma vitellogenin levels (Carragher et al.,
1989), and to inhibit vitellogenin synthesis in tilapia
hepatocytes (M.M. Vijayan and A. Takemura, unpublished data), and it brings about a 30% decrease in
liver oestradiol binding capacity (Pottinger and Pickering, 1990) together with a transient (peaking at two
weeks) one-third increase in plasma oestradiol binding
ability. Although no effects on total plasma oestradiol are noted, the combination of increased plasma
oestradiol binding capacity and decreased liver oestradiol binding will substantially reduce the biological

256
actions of the sex steroid. Thus cortisol interferes
with reproductive function in immature and maturing rainbow trout (Chakraborti et al., 1987), reiterating a phenomenon described above for embryos
exposed to cortisol (Van den Hurk and Van Oordt,
1985).

Future directions
Even if we ignore stress as a guiding principle, there
are so many unresolved aspects of cortisol action in
fishes that it seems a little overwhelming to pick specific areas for future directions. Naturally, the few
topics mentioned below will reflect the authors bias,
including that towards physiological effects. Experimentally, the approaches are muddled by a number of
disconcerting facts, not least by the excitability of the
cortisol system and the apparent extreme sensitivity
of some parameters to minor changes in endogenous
cortisol. The underlying diurnal and seasonal cycles
in plasma cortisol and cycles associated with feeding,
smoltification and sexual maturation add further levels
of complexity. In fact, it appears that even the slightest
experimental manipulation is likely to alter the cortisol
set-point, making it a difficult task, posthoc, to identify
cortisols elusive housekeeping actions. However, a
combination of molecular approaches, isolated cell
systems and whole-fish physiology should allow an
improved understanding of the many faces of cortisol.
Identification of GREs in enzyme genes will help
identify natural targets for cortisol action, and analyses
of expression and turnover of these targets will provide
some indication of the fine-tuning exerted by cortisol
alone and in combination with other hormones. Work
on isolated cell systems or immortalized cell lines
will make it possible to isolate cortisol actions from
those of other hormones and to obtain a first glimpse
of potential interactions with other hormones. Irrespective of their noted shortcomings, compounds such
as metyrapone, RU 486 and dexamethasone will be
extremely useful to better define cortisol actions on a
whole-animal level.
Conceptually, a few interesting areas will have to
be cleared up before it will be possible to approach
interesting evolutionary questions. For instance, the
apparent presence/absence of cortisol plasma-binding
protein needs to be established conclusively and the
consequences for cortisol transport and tissue uptake
need to be analysed, together with an estimation of
biologically available concentrations of cortisol. Bet-

ter information regarding how cortisol travels and

how the active fraction is regulated is essential to
the understanding of hormone/receptor interactions
as is receptor expression, posttranslational processing
of the receptor itself and covalent regulation. In the
context of the GR, the absence of a distinct mineralocorticoid in the fishes remains an enigma with
interesting ramifications as to the nature and evolution
of the unique (?) cortisol receptor.
The search for nongenomic actions of the hormone
should be continued. Careful, short-term experimentation is required and should involve an analysis of the
role of protein kinases, intracellular calcium transients
and interactions with nonsteroid hormones for which
intracellular message transduction systems are known
or at least better defined. In this framework, RU 486,
dexamethasone and inhibitors of protein and nucleotide biosynthesis provide valuable tools to separate
genomic from nongenomic actions. For the genomic
actions, identification of potential permissive effects
would make an interesting contribution to the functional evolution of the cortisol/glucagon system and
would provide clues as to interactions with insulin and
the IGF system.
A causality needs to be established for the many
parameters for which a correlation with plasma
cortisol has been noted. At the receptor level, the
importance of different hsps to glucocorticoid receptor
function needs to be elucidated. The wide temperature range experienced by fishes and the many natural
environmental effectors we hesitate to use the term
stressors should superimpose an interesting layer
of hsp dynamics on their potential interaction with
the GR. Identification of GREs in some of the obvious targets of cortisol actions should be a priority.
These include the promoters for hormones such as
insulin, glucagon, IGF-1 and not least, growth hormone. Similarly, an analysis of the promoters for some
of the cortisol-responsive enzymes listed in Tables 5
to 8 could serve as a promising hunting ground for
GREs in fishes, keeping in mind the close relationship
between consensus in GREs and oestrogen-responsive
elements in other vertebrates and the identification of
several potential GREs in the trout oestrogen-receptor
gene already (Teitsma et al., 1998).
One of the least experimentally accessible tissues,
yet most interesting in the context of cortisol, is the
white muscle. Because of its exceptional bulk in our
finned friends, the tissue may impart its metabolic
imprint on the rest of the fish, even if changes at
the muscle level may straddle the threshold of detect-

257
ability. Although glucocorticoid-receptor activity in
muscle is barely measurable (Table 3), the tissue
is likely to contain higher numbers of corticosteroid receptors than any other tissue in the fishs body,
but we are still unaware how these receptors interact with the ligand or how the hormone works. With
the mechanisms of glucocorticoid-driven proteolysis
currently being probed in mammals (Auclair et al.,
1997), and its immense metabolic implications in
fishes, the interplay of cortisol with muscle is likely to
open a fascinating conceptual window on the multifaceted and important roles of this powerful hormone in
fishes.

Summary
In addition to the relatively well-defined actions during stress, cortisol is a major regulator of intermediary metabolism and normal physiological parameters in fishes. In this review, we cover biosynthesis,
degradation, turnover and transport of cortisol, before
detailing different stages and levels of cortisol action.
Starting with molecular and binding characteristics of
the glucocorticoid receptor, we detail the interactions
between cortisol and its receptor, then focus on the
abundance and the role of cortisol in the early life
history of fishes. Distinguishing between genomic and
nongenomic modes of action, we analyse the varying
effects of the hormone on carbohydrate and lipid metabolism, including gluconeogenesis, and peripheral
and hepatic lipolysis. The multifaceted actions of the
hormone on amino acid and protein metabolism are
discussed in the framework of proteolysis and biosynthesis of key enzymes of glycogen turnover, pentose
shunt, and amino acid turnover. The problems of
manipulating endogenous concentrations of cortisol in
fishes are discussed in the context of the usefulness of
metyrapone, RU 486 and dexamethasone. We briefly
discuss interactions of cortisol with other hormones
in fishes, such as insulin and growth hormones, and
the role cortisol may play in the response to xenobiotics and involvement of the hormone in the regulation
of growth and reproduction. Stress-related action and
the function of cortisol in smoltification and hypoosmoregulation are touched upon, but do not form
the focus of this review. It is clear that even though
the literature is extensive with respect to cortisol and
metabolism, we are a long way from a complete
understanding of the role of this complex hormone in
fish.

Acknowledgements
The authors gratefully acknowledge support from the
Natural Sciences and Engineering Research Council
(NSERCCanada). We appreciate Dr Charles Hollingworths encouragement and patience throughout the
writing of this review.

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