ARTÍCULO ESPECIAL
When glucocorticoids change from protective to harmful. Lessons from a type 1 diabetes animal model*
Yanina Revsin, E. Ronald de Kloet
Department of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research (LACDR) and Leiden University Medical Center (LUMC), Leiden University, The Netherlands
Postal address: Dr. E.Ronald de Kloet, Department of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research (LACDR) & Leiden University Medical Center (LUMC), Gorlaeus Laboratory, PO Box 9502, 2300 RA Leiden, The Netherlands Fax (0031) 71-5274715 e-mail: e.kloet@lacdr.leidenuniv.nl
* Presented in part at the SAIC Meeting, Mar del Plata, 23 November 2007.
Abstract
A fundamental question in the neuroendocrinology of stress and adaptation is how stress mediators
that are crucial for resilience and health can change into harmful signals enhancing vulnerability to
disease. To address this question we focus in the rodent on corticosterone as end product of the hypothalamicpituitary-
adrenal (HPA) axis, which coordinates the behavioural and physiological response to stressors. The action
of corticosterone is mediated by mineralocorticoid (MR) and glucocorticoid receptors (GR). The receptors are
transcription factors regulating gene transcription but recently these nuclear receptors were found to mediate
also rapid non-genomic actions. MR participates in the initial stress reaction important for appraisal and coping
processes, while management of the later adaptive phase primarily depends on GR. Imbalance in stress mediators
is a characteristic feature of a phenotype vulnerable for stressors. This concept calls for recovery of the MR:GR
balance as a therapeutic strategy to promote resilience still present in the diseased brain. As an example, we
discuss in this article, how the impact of excessive levels of corticosterone in a pharmacological model of type 1
diabetes can be ameliorated after a brief treatment with a GR antagonist.
Key words: Diabetes; Stress; Stress hormones; Glucocorticoids; Glucocorticoid receptors; Molecular markers
Resumen
Cuando los glucocorticoides cambian de protectores a dañinos en un modelo animal de diabetes tipo 1. Una cuestión fundamental en la neuroendocrinología del estrés y la adaptación es
saber cómo los mediadores del estrés cruciales para la resiliencia y la salud pueden convertirse en señales
dañinas que aumentan la vulnerabilidad a las enfermedades. Para responder a esta pregunta nosotros nos
centramos en la corticosterona como producto final del eje hipotálamo-hipófisis-glándula adrenal de los ratones,
que coordina las respuestas fisiológicas y conductuales hacia los agentes estresantes. La acción de la
corticosterona es mediada por los receptores de los mineralocorticoides (MR) y glucocorticoides (GR). Estos
receptores son factores nucleares que regulan la transcripción de los genes aunque también se ha encontrado,
hace poco, que pueden ser mediadores de acciones extra genómicas rápidas. Los MR participan en la respuesta
inicial al estrés con relevancia en la evaluación y enfrentamiento al agente estresante, mientras que la fase
adaptativa tardía depende de los GR. El desequilibrio de estos mediadores del estrés es rasgo característico de
un fenotipo vulnerable a los agentes estresantes. La idea es recuperar el equilibrio MR:GR como una estrategia
terapéutica para promover la resiliencia aún presente en el cerebro enfermo. Como ejemplo discutimos en este
artículo cómo, en un modelo farmacológico de diabetes tipo 1, el impacto de niveles excesivos de corticosterona
puede ser atenuado por un breve tratamiento con antagonistas de los GR.
Palabras clave: Diabetes; Estrés; Hormonas del estrés; Glucocorticoides; Receptores para glucocorticoides; Marcadores moleculares
Any stressor -either real or imagined- that threatens to disturb homeostasis triggers a physiological and behavioural response aimed to promote adaptation. The adaptation to the stressor is coordinated in body and brain by humoral and nervous systems of which the hypothalamic- pituitary-adrenal (HPA) axis and the sympathetic nervous system (adrenaline) are the primary mediators (Fig. 1). The end product of the HPA axis: cortisol (human) and corticosterone (human, rodents), collectively called CORT here, does not act alone but operates in concert with the humoral and nervous signals that mediate the stress response1. While CORT is essential for health, the hormone becomes damaging if its circulating levels are either excessive or inadequate to deal with the stressor. A fundamental question in the biology of stress is therefore how CORT can change from a protective into a harmful signal.
Fig. 1.- Brain regions activated after stress exposure (Amy;
amygdala, Hipp: hippocampus, PFC: prefrontal cortex) and
output of these areas through the hypothalamus (HYP). A
resulting activation of the fast acting sympatho-adrenomedullar
system (right) and the slower acting HPA axis
system (left) will affect the function of peripheral organs
and feedback to the brain via adrenaline and corticosterone,
respectively. Adrenaline will eventually activate
noradrenergic transmission from the locus coeruleous (LC),
reaching the amygdala, prefrontal cortex and hippocampus
among other areas. Corticosterone readily penetrates
the blood-brain barrier and acts on receptor containing
brain areas such as the limbic brain structures and the
paraventricular nucleus in the hypothalamus. SNS: sympathetic
nervous system; ACTH: adrenocorticotropin hormone;
CRH: corticotrophin releasing hormone. Reprinted
with permission from Joels and Krugers, Neuronal Plasticity,
2007.
How does CORT act in the stress response? CORT
dampens the initial stress (defence) reactions to the stressor,
and prevents them from overshooting2. This action is
mediated by the receptors that control gene transcription
and has as wide a diversity as the variety of stressors.
However, the very same CORT actually also can boost
the initial stress reaction via newly discovered membrane
receptors for the hormone in the hippocampus that can
enhance excitatory neurotransmission3, 4. Moreover,
CORT can mobilize the energy needed to perform these
costly regulations of the stress response, and store energy
for future use when coping with stress is successful.
The secretory pattern of CORT has been used widely
as marker for health and disease. CORT is secreted under
basal conditions in hourly pulses which are increased
in amplitude during the activity period, i.e. night time in
rodents and daytime in man. During stress-related disease
and aging the patterns become disordered, a sign
that the HPA axis pulsatility has become dysregulated5.
CORT secretory bursts can be triggered by a stressor
any time. Like the basal pattern also the stress-induced
CORT pattern serves as a biomarker for breakdown of
adaptation and stress-related disease. A healthy resilient
organism is characterized by a rapid activation of the
CORT as long as the secretion of the hormone is also
turned off efficiently.
During the stress response CORT and the other HPA
axis hormones mediate the ability to cope. For this purpose
CORT targets in the brain precisely those circuits
that have perceived the initial trigger of the stress response;
the action exerted by the hormone can be divided
in two temporal domains: a fast activating and a
slower suppressive phase (Fig. 2). These actions are
mediated by two types of corticosteroid receptors: mineralocorticoid
receptors (MR) and glucocorticoid receptors
(GR)6, 7. MR and GR are nuclear receptors operating as
transcription factors. MR is abundantly expressed in limbic
structures involved in mood, affect and memory processes.
GR has is expressed widely in neurons and glial
cells with highest abundance in centers involved in regulating
the stress response and also in limbic structures.
Thus, in limbic structures such as e.g. the neuronal circuits
of hippocampus (CA1-2 and dentate gyrus), the
amygdala nuclei and areas of the prefrontal cortex MR
and GR are co-localized and expressed in large amounts6.
Fig. 2.- Time course of molecular, cellular and behavioral responses
to stress hormones. MR: mineralocorticoid
receptors, GR: glucocorticoid receptors. AVP: arginine
vasopressin. Reprinted with permission from de Kloet,
Joels, and Holsboer, Nature Reviews Neuroscience, 2005.
An important target of glucocorticoids is the hippocampus, where GR and MR exert in a co-ordinate manner complementary actions of the glucocorticoids. It is thought that the activating MR and the suppressing GR need to operate in balance to control homeostasis and health. Thus, imbalance in the MR:GR ratio enhances susceptibility to disease for which the individual is predisposed. Currently, drugs are being developed that restore MR:GR imbalance with the purpose to correct stress-related dysregulations causal to disease. Here we will illustrate this concept with an animal model for diabetes, which is characterized by aberrant actions of CORT that can be corrected by a CORT antagonist.
Diabetes as a disease model for aberrant CORT
Type 1 diabetes (T1D) complications, such as peripheral
and autonomous neuropathy, are well-recognized. However,
T1D can also impact the integrity of the central nervous
system (CNS) as appeared from the pioneering studies
of De Nicola and colleagues at the Instituto de Biología
y Medicina Experimental in Buenos Aires (IBYME-CONICET)
8. How T1D affects CNS integrity remains to
be elucidated. High association of T1D and psychiatric
disorders, notably depression, were reported in crosssectional
studies9. The underlying mechanism and the
causality behind this association still need to be identified.
A direct adverse effect of diabetes leading to psychiatric
disorders, especially depression, was reported by
Lustman et al10 and alterations in the activity of the HPA
axis were reported in diabetic patients11. In addition, studies
on diabetic patients demonstrated mild to moderate
slowing of mental speed and diminished mental flexibility12.
Although the alterations in cognitive functions under
normal conditions are not severe, mild cognitive defects
can influence everyday activities in more demanding situations.
While the mechanism that underlies this cognitive
impairment is poorly understood, Sandeep et al reported
in 200413 that hypercortisolism in diabetic patients may
contribute to the dysfunction of the hippocampus, a brain
area that belongs to the limbic system and plays major
roles in memory processes and spatial navigation.
To investigate disease initiation, progression, and treatments
without exposing humans to unnecessary and potentially
unethical risks, animal models have been developed.
They have contributed important knowledge regarding
the study of diabetes. The physiology of mice, rats,
and other animals is remarkably conserved in comparison
to the human condition, and over the last 40 years
several models have become available. For our studies
we have used two animal models, i.e. a pharmacological
model, the streptozotocin (STZ)-treated mouse, and a
genetic model, the non-obese diabetic (NOD) mouse,
which spontaneously develops the disease. Like type 1
diabetic patients, these animal models show high circulating
levels of glucocorticoids, increased sensitivity to
stressors, and morphological alterations in various brain
areas14-16. Our results obtained from experiments with the
STZ model will be described here.
When T1D develops, the lack of insulin causes both
hyperglycemia and cellular starvation, a condition known
as metabolic stress. The metabolic stressor, as any other
kind of stress condition, activates the HPA axis leading to
increased corticosterone levels and occupancy of GRs in
brain and pituitary. Previous reports in T1D animal models
showed GR downregulation in the hippocampus17 and
HPA axis hyperactivity18. In addition, elevated glucocorticoids
further enhance the hyperglycemic effect of insulin
by inhibiting cellular glucose uptake, also in neurons
and glial cells in the brain. Therefore, hyperglycemia will
become chronic. In this way, continuous metabolic alterations
will contribute to the defective shut-off of the stress
response. During chronic hypercorticism various pathophysiologies
are promoted including dysfunction of the
hippocampus19, 20. Remodelling of the structure of the
hippocampus is accelerated and its vulnerability to cognitive
dysfunctions is enhanced.
A fundamental question in the neuropathophysiology
of T1D is therefore whether glucocorticoids aggravate the
functional and morphological signs of neurodegeneration
and cognitive impairment. In our studies we tested the
hypothesis that the onset of diabetes induces dysregulation
of the HPA axis and hypersecretion of glucocorticoids
which then renders the brain more vulnerable to
metabolic insults causing damage and concomitant cognitive
disturbances.
The STZ-induced diabetes mouse model showed that
the primary event leading to hypercorticism is hyperresponsiveness
of the adrenal gland21. To further investigate
the underlying mechanism leading to the observed
hypercorticism in diabetes we analyzed molecular markers
of the HPA axis activity at different time points. The
results showed a profound dysregulation of the HPA axis
and a time-dependent adaptation to the new metabolic
condition. Whether this adaptation in T1D leads to a more
fragile state of the brain in which glucocorticoid excess
may enhance the potential for damage and attenuate a
protective mechanism, thus facilitating cognitive impairment
was addressed in the next two experiments.
First, the hippocampus of the diabetic mice exhibited
increased neuronal activation, signs of oxidative stress
and astrogliosis. These data support the concept that
uncontrolled diabetes produces signs of hippocampal
pathology, which procedes together with changes in other
brain structures such as hypothalamus and cerebral cortex16,
21. Cognitive deficits were observed in the hippocampus-
dependent novel object-placement recognition
(NOPR) task. The NOPR task uses the spontaneous exploration of objects in a novel environment22 and thus allows
the study of mild hippocampal alterations. Non-diabetic
control mice preferred the exploration of the object
placed in a novel location while the diabetic mice do not.
This reduced exploration of the diabetic mice indicates
impaired spatial object-placement memory23, 24 (Fig. 3).
Interestingly, performance in the socalled Morris water
maze, retention in the forced swim task and emotional
parameters in the elevated plus maze were not affected
in diabetic mice. These tasks possibly are insensitive to
the moderate hippocampal disturbances observed at early
stages of diabetes, while during a more prolonged disease
state behavioral deficits were established24.
Fig. 3.- Mifepristone (a glucocorticoid receptor antagonist) effect on the novel
object-placement recognition (NOPR) task. A) Photograph of the NOPR task:
during the sample trial the two objects (water bottles) are equally explored
(data not shown). After 50 minutes, during the test trial, one of the objects is
re-located. B) Percentage of preference for the re-located object during the
test trial for control and 11-days diabetic mice vehicle- and mifepristonetreated.
Values expressed mean ± SEM. *p<0.05.
Second, to clarify the role of hypercorticism in the hippocampus
of STZ-diabetic mice we have used
mifepristone (RU38486), an antagonist of the action of
glucocorticoids at the GR. We showed that the continuous
blockade of glucocorticoid action by treatment with
mifepristone for 4 consecutive days (from day 6 to 10 of
after onset of diabetes in the STZ model) prevented some
of the hippocampal aberrations and reversed others24. The
prevention of astrogliosis and excessive neuronal activation
in the hippocampus showed that mifepristone treatment
can interfere directly with the progression of hippocampal
alterations observed in diabetic mice24, 25. Cognitive
deficits observed at day 11 of diabetes were ameliorated.
Surprisingly, the diabetic animals treated with
mifepristone performed even better than the untreated
controls. A rationale behind this observation is that the
blockade of the GR would allow a more prominent function
of neuroprotective MR-mediated actions26. Hence this
would predict that during GR blockade in the face of high
circulating glucocorticoids the maintenance of hippocampal
integrity is a necessary condition for improved performance
in the NOPR task 27.
In addition to this role of glucocorticoids in diabetes
neuropathology other factors altered in T1D might also
be involved. For instance, the imbalance of glucose metabolism
may be of importance in modulating brain disturbances
induced by diabetes. Some studies have described
that hyper- and hypoglycemic episodes can
cause acute cerebral dysfunction in diabetic animals14,
28. In the STZ-induced mouse model of T1D, GR antagonist
normalized hippocampal functions regardless
the hyperglycemic state, which is not altered in diabetic
mice treated with mifepristone. Therefore, it is unlikely
that hyperglycemia per se accounts for the hippocampal
disturbances observed. These findings are in agreement
with a recently published report22, in which corticosterone
replacement of adrenalectomized rats at
physiological concentrations restored hippocampal functions,
while glycemia remained elevated. However, the
possibility of an additional effect of glucocorticoid excess
on hippocampal glucose metabolism in T1D can
not be discarded.
Furthermore, cerebral dysfunction in T1D can also be
a result of insulin deficiency. Although several studies
describe a central role of insulin in animal models of diabetes29-31, a recent report revealed that hippocampal impairments
are not determined by changes in insulin production22.
Nevertheless, it is likely that the negative effect
of diabetes on hippocampal plasticity may be attributable
to an interaction between elevated glucocorticoids and
insulin receptor signaling.
In conclusion, the results described in this article provide
evidence that glucocorticoid excess and the concomitant
continuous activation of the GR are responsible for
molecular changes and hippocampal-associated behavioural
dysfunction at the early stages of diabetes. They
indicate that in T1D excess glucocorticoids have lost their
function in restoring homeostasis and have become damaging
to the brain. The receptors for glucocorticoids may
therefore be an excellent target for a therapy aimed to
normalize the disturbed hippocampal functions characteristic
for diabetes neuropathology.
Acknowledgements: The support of the Netherlands Science Foundation-WOTRO (NWO-WOTRO project #88-252) dedicated to collaboration with Prof AF de Nicola's research group at the Instituto de Biología y Medicina Experimental in Buenos Aires (IBYME-CONICET), the Leiden-Trier International Research Training Group (IRTG) of the Deutsche Forschungs Gemeinschaft (DFG) and NWO (project #NWODN-95-420) and the Royal Netherlands Academy of Art and Sciences (KNAW, Academy professorship to E.R. de Kloet) is gratefully acknowledged.
Conflicts of interests: E.R. de Kloet is member of the Scientific Advisory Board of Corcept Therapeutics Inc.
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Received: 18-2-2009
Accepted: 16-3-2009