(Received for publication, October 12, 1995)
From the
Peroxisome proliferator-activated receptors (PPARs) are nuclear
hormone receptors that can be activated by fatty acids and peroxisome
proliferators. The PPAR subtype mediates the pleiotropic effects
of these activators in liver and regulates several target genes
involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR
gene is regulated at the
transcriptional level by glucocorticoids. We investigated if this
hormonal regulation also occurs in the whole animal in physiological
situations leading to increased plasma corticosterone levels in rats.
We show here that an immobilization stress is a potent and rapid
stimulator of PPAR
expression in liver but not in hippocampus. The
injection of the synthetic glucocorticoid dexamethasone into adult rats
produces a similar increase in PPAR
expression in liver, whereas
the administration of the antiglucocorticoid RU 486 inhibits the
stress-dependent stimulation. We conclude that glucocorticoids are
major mediators of the stress response. Consistent with this hormonal
regulation, hepatic PPAR
mRNA and protein levels follow a diurnal
rhythm, which parallels that of circulating corticosterone. To test the
effects of variations in PPAR
expression on PPAR
target gene
activity, high glucocorticoid-dependent PPAR
expression was
mimicked in cultured primary hepatocytes. Under these conditions,
hormonal stimulation of receptor expression synergizes with receptor
activation by WY-14,643 to induce the expression of the PPAR
target gene acyl-CoA oxidase. Together, these results show that
regulation of the PPAR
expression levels efficiently modulates
PPAR activator signaling and thus may affect downstream metabolic
pathways involved in lipid homeostasis.
The peroxisome proliferator-activated receptors (PPARs) ()are orphan nuclear hormone
receptors(1, 2) . To date, three different subtypes,
PPAR
, PPAR
or -
(also named FAAR or NUC1), and
PPAR
, have been cloned in amphibians(3) ,
rodents(4, 5, 6, 7, 8, 9, 10) ,
and man(11, 12, 13) . Within the nuclear
hormone receptor superfamily, PPARs belong to the subfamily that
comprises the thyroid hormone receptors and retinoic acid receptors (14) . The first PPAR cDNA cloned was isolated from a mouse
liver library and corresponds to the PPAR
subtype(4) .
This receptor was shown to be activated by peroxisome
proliferators(3, 4) , a class of compounds that have
characteristic pleiotropic effects, especially in rodents. In
hepatocytes, peroxisome proliferators cause a dramatic increase in the
number and the size of peroxisomes, an effect associated with a
concomitant induction of the activity of several enzymes of the
peroxisomal
-oxidation pathway(15) . Definitive proof that
PPAR
is the major mediator of these effects was provided by the
absence of the typical hepatic response to peroxisome proliferators in
PPAR
-deficient mice generated by targeted disruption of the
PPAR
gene(16) . Whereas little is known about the
mechanisms that lead to peroxisome proliferation, the associated
increase in the level of expression of several genes is better
understood. Indeed, several PPAR
target genes were identified that
contain one or several PPAR-responsive elements in their
promoter(1, 2) . To date, the known PPAR
target
genes code for enzymes involved in the following metabolic pathways:
(i) activation of fatty acids to acyl coenzyme A (CoA) derivatives
(acyl-CoA synthetase, (17) ), (ii) peroxisomal
-oxidation
(acyl-CoA oxidase (ACO), Refs. 3, 18, and 19, and bifunctional enzyme, (20) ), (iii) mitochondrial
-oxidation (medium chain
acyl-CoA dehydrogenase, (21) ), (iv) microsomal
-oxidation (CYP 4A6, (22) ) and (v) ketogenesis
(hydroxymethylglutaryl-CoA synthase, 23). In addition, the genes coding
for apolipoproteins AI(24) , AII(25) , and CIII (26) are also regulated by PPAR
, suggesting an involvement
of these receptors in the regulation of the extracellular transport of
lipids.
Although peroxisome proliferators are now used as
prototypical activators of PPAR, there is still no evidence that
these compounds bind directly to the receptor. The only PPAR ligands
identified so far are antidiabetic agents of the thiazolidinedione
family, which bind with high affinity to PPAR
(27) . The
discovery that several fatty acids, such as arachidonic acid or
linoleic acid, activate PPAR
suggests that fatty acids could
represent biological activators(5, 18) . According to
this hypothesis, PPAR
could function as a fatty acid sensor, which
would allow the fatty acids to regulate their own
metabolism(1, 2) .
PPAR mRNA is predominantly
expressed in tissues capable of oxidizing fatty acids, such as brown
adipose tissue, liver, heart, kidney, and muscle(8) . Absence
of PPAR
expression in knockout mice prevents the inducibility of
several target genes in liver, including ACO and bifunctional enzyme,
by peroxisome proliferators, suggesting that the level of expression of
PPAR
is important for the proper regulation of these genes in
vivo(16) . In fact, PPAR
expression in adult rat
liver is subject to marked interindividual variations for so far
unknown reasons. (
)We and others (28, 29) have shown recently that PPAR
expression
is directly regulated at the transcriptional level by glucocorticoids
in rat hepatocytes or hepatoma cell lines cultured in vitro.
This regulation is mediated by the glucocorticoid receptor and does not
involve stabilization of the mRNA(28) . These findings
suggested that PPAR
expression in liver could be subject to
hormonal regulation in vivo in situations of high circulating
glucocorticoid levels.
In the present study, we show that PPAR
mRNA levels increase in rat liver during an immobilization stress
situation and follow a diurnal rhythm. The impact of variations in the
levels of PPAR
expression on the regulation of a prototypical
PPAR
target gene, the ACO gene, is furthermore analyzed using
hepatocytes in primary culture.
Figure 1:
Stress
stimulates PPAR expression in liver. A 4-h immobilization stress
was achieved as described under ``Materials and Methods.''
Total RNA (15 µg) was analyzed by RNase protection assay using a
probe for the PPAR
mRNA and a probe for the mRNA of the large
ribosomal subunit 27-kDa protein (L27) as control. A, in the
liver (left panel), the stressed animals (S, n = 3) show higher levels of PPAR
mRNA than the
unstressed control animals (C, n = 3). In
contrast, there is no variation in PPAR
mRNA levels in the
hippocampus of the same animals (right panel). B,
graphic representation of PPAR
mRNA levels in liver and
hippocampus of the control (C) and stressed (S)
animals. PPAR
mRNA levels were normalized to those of L27. The
PPAR
mRNA level in liver was arbitrarily set to 1. C,
plasma corticosterone (CS) levels (ng/ml) of stressed (S) and
control animals (C). Results are the mean ± S.D. of
three animals.
To test whether
glucocorticoids are indeed involved in the stress-dependent stimulation
of PPAR expression, animals were treated before immobilization
with the specific glucocorticoid antagonist RU 486 (30 mg/kg BW), or
saline as control, and the effect on PPAR
mRNA levels was
analyzed. In the saline-injected animals, the 4-h immobilization
produced a significant 3-fold increase of PPAR
mRNA levels in
liver (Fig. 2). In marked contrast, the stress-dependent
induction of PPAR
expression was inhibited in the rats injected
with RU 486 (Fig. 2). These results demonstrate that
glucocorticoids are the major endocrine mediators of the induction of
PPAR
expression by stress.
Figure 2:
RU 486 inhibits the stress-dependent
stimulation of PPAR expression. Rats were injected subcutaneously
either with vehicle (SHAM) or with 30 mg/kg BW RU 486.
Immediately after the injection, the control animals (C) were
returned to their cage, whereas the stressed animals were subjected to
a 4-h immobilization. Liver total RNA was analyzed as described in Fig. 1. A, representative results obtained after
vehicle (SHAM) or RU 486 injection in control (C) and
stressed (S) rats are shown. B, quantification of
L27-normalized PPAR
levels in liver of vehicle (SHAM) or
RU 486 injected stressed (S) and control (C) animals.
Results depict the mean ± S.D. of three
animals.
If glucocorticoids are indeed
directly involved in the stimulation of PPAR mRNA expression
induced by stress, the acute injection of exogenous glucocorticoids
should lead to increased PPAR
mRNA levels. Rats were hence
injected either with saline or with dexamethasone (40 µg/kg BW) and
sacrificed 4 h later. As predicted, dexamethasone-injected rats
displayed 3.5-fold higher PPAR
mRNA levels in liver as compared
with saline-injected animals (Fig. 3, A and B). Thus, a single injection of the glucocorticoid agonist
dexamethasone reproduced the effects of endogenous glucocorticoids
secreted in response to stress. Another effect of administration of
dexamethasone was the well described blockade of the
hypothalamo-pituitary-adrenal axis(37) , resulting in a
dramatic decrease of the levels of circulating corticosterone (Fig. 3C).
Figure 3:
Dexamethasone induces PPAR expression
in liver. Rats were injected intraperitoneally either with vehicle (C) or 40 µg/kg BW dexamethasone (DEX). The
animals were sacrificed 4 h later. A, representative results
obtained after vehicle (C) or dexamethasone (DEX)
injection. B, quantification of L27-normalized PPAR
levels in liver of vehicle (C) or dexamethasone (DEX)
injected animals (n = 3). C, plasma
corticosterone levels (ng/ml) 4 h after vehicle (C) or
dexamethasone (DEX) injection. The mean ± S.D. of three
animals are shown.
Figure 4:
Diurnal variations of PPAR mRNA and
protein levels in liver. Animals were sacrificed at one time point in
the morning (9:30 a.m., n = 3) and three time points in
the afternoon (3:30 p.m., 5:00 p.m., and 6:30 p.m., n =
3 each). For each time point, the three animals were sacrificed during
three consecutive days. A, plasma corticosterone levels
(ng/ml) at each time point (n = 3). B,
L27-normalized PPAR
mRNA levels at each time point (n = 3), determined as described in the legend to Fig. 1. C, PPAR
protein levels in liver nuclear
extracts as analyzed by Western blotting. The affinity-purified
anti-PPAR
antibody detects a protein of about 55 kDa corresponding
to the PPAR
protein (arrow). This signal is not detected
when preimmune IgGs are used (PI, lane 1, same extract as in lane 4) and is markedly reduced when the antibody is
co-incubated with 20 µg of the purified GST-PPAR
/AB fusion
protein (lane 6, same extract as in lane 4). Lanes 2-5 correspond to liver nuclear extracts from the
animals sacrificed on day 2 (see D) at 9:30 a.m., 3:30 p.m.,
5:00 p.m., and 6:30 p.m., respectively. Unspecific bands are indicated
with asterisks. D, the L27-normalized PPAR
mRNA
levels (solid line,
) and protein levels (dashed
line,
) are plotted successively in the sequence the
animals were killed during the 3 days of the experiment. PPAR
mRNA
and protein levels at 9:30 a.m. were set to 1 arbitrary unit (A.U.). Results in A and B represent the
mean ± S.D. of three animals.
To test whether the diurnal variations of PPAR
mRNA levels resulted into changes in PPAR
protein expression, the
relative levels of the receptor were measured in liver nuclear
extracts. PPAR
protein was detected on Western blots using an
anti-PPAR
antibody. This antibody detects a major band at 55 kDa,
which corresponds to the predicted size of PPAR
. This signal is
specific, since it is not detected by preimmune IgGs (Fig. 4C, lane 1). Moreover, its intensity is
markedly reduced when the antibody is co-incubated with 20 µg of
the purified antigen (Fig. 4C, lane 6). In
nuclear extracts from the liver of the animals analyzed during the
second day of the 3-day experiment, PPAR
protein levels were low
in the morning and 2-, 3-, and 5-fold higher at 3:30 p.m., 5:00 p.m.,
and 6:30 p.m., respectively (Fig. 4C, lanes
2-5). Similar results were obtained for the two other days
of the experiment. Thus, when the levels of PPAR
mRNA and protein
measured in each individual animal are plotted successively, according
to the time at which the animal was sacrificed, both mRNA and protein
levels show a striking cyclic pattern of expression over the three
consecutive days (Fig. 4D).
Figure 5:
Dexamethasone potentiates the induction of
ACO gene expression by WY-14,643. A, rat hepatocytes in
primary culture were treated during 6 h in presence or absence of 1
µM dexamethasone (DEX). Nuclear extracts were
analyzed by Western blotting as described in the legend to Fig. 4C. The position of PPAR protein is indicated (arrow). An unspecific band is indicated (asterisk). B, hepatocytes in primary culture were treated during 24 h
with 1 µM dexamethasone (lanes 3 and 4)
and 100 µM WY-14,643 (lanes 2 and 4).
ACO, PPAR
, and L27 mRNA were detected by RNase protection as
described in the legend to Fig. 1. C, L27-normalized
ACO mRNA levels. The ACO mRNA level of the vehicle-treated hepatocytes
was arbitrarily set to 1. F.I. = -fold
induction.
The hormonal response to stress involves essentially the
release of catecholamines by the sympathetic nervous system and the
secretion of glucocorticoids by the adrenal medulla through the
activation of the hypothalamo-pituitary-adrenal axis(35) . The
onset and the duration of the glucocorticoid component of the stress
response are slower and more sustained, respectively, than those of the
catecholamine component. Thus, elevated glucocorticoid levels can be
considered as a second hormonal wave following the initial peak of
catecholamines. Our results demonstrate that, in vivo, the
PPAR gene responds mainly to the glucocorticoid component of the
hormonal response to stress. Indeed, using the antagonist RU 486 and
the agonist dexamethasone, it was shown that glucocorticoids are
necessary and sufficient to induce PPAR
gene expression in liver
during stress situations. Interestingly, stress was unable to modify
PPAR
expression in the hippocampus, despite the presence of
glucocorticoid receptor in this tissue. One hypothesis is that
liver-specific factors are required to permit the regulatory action of
glucocorticoid receptor on the PPAR
gene promoter. Alternatively,
brain-specific factors might inhibit this regulation. In liver, the
induction of PPAR
mRNA is a fast response since it can be observed
already 4 h after immobilization or agonist injection. The rapid
regulation of PPAR
mRNA expression in vivo reported
herein argues for a similar direct transcriptional effect of
glucocorticoids on PPAR
gene expression in liver as in hepatocytes
cultured in vitro(28, 29) .
The metabolic
response to stress is characterized by energy mobilization. Under the
action of lipolytic hormones (catecholamines mainly), fatty acid
mobilization occurs in the adipose tissue. In liver, the mobilized
fatty acids enter the -oxidation pathway and ketogenesis is
stimulated(42) . Remarkably, PPAR
regulates genes involved
in the activation of fatty acids as well as in the
-oxidation and
ketogenesis pathways(1) . Thus, stimulation of PPAR
expression in liver by glucocorticoids during stress may potentiate the
regulation of these target genes and contribute to the stimulation of
the metabolic pathways involved in energy homeostasis.
The
expression of the PPAR gene is showing a diurnal cycling pattern
in liver, which parallels that of circulating corticosterone. This is
consistent with a high sensitivity in liver of the PPAR
gene to
the levels of circulating corticosterone. The diurnal variations of
PPAR
mRNA is closely followed by a parallel cycling of PPAR
protein suggesting that PPAR
mRNA is efficiently translated.
Furthermore, the cyling of the PPAR
protein levels implies that
the half-life of the protein is short enough to allow its levels to
significantly decrease after 12 h. Altogether, these results suggest
that PPAR
signaling pathway may be efficiently modulated by a
rapid and transient regulation of receptor levels. Post-translational
mechanisms may furthermore exist, since we did not detect clear
variations of PPAR
protein levels after a 4-h stress (data not
shown). Alternatively, this very short time of stimulation may be
insufficient to give rise to a detectable increase of PPAR
protein
levels.
The investigation of the glucocorticoid-dependent regulation
of PPAR gene was possible in the whole animal, since physiological
situations associated with variations of circulating glucocorticoids
are well characterized. Moreover, specific glucocorticoid agonists and
antagonists are available. More difficult is the in vivo study
of PPAR
-mediated gene regulation, since the natural ligands of
this receptor are still unknown, and specific agonists or antagonists
have not been reported so far. The PPAR activators known to date, such
as peroxisome proliferators, have pleiotropic effects in vivo,
making it difficult to discriminate between the direct and indirect
actions of these drugs in the animal. In contrast, hepatocyte primary
cultures represent a model close to the in vivo situation,
which is, however, isolated from the hormonal and metabolic complexity
of the intact animal. Using this system, we show that high levels of
expression of PPAR
and activation of the receptor are necessary
for a maximal stimulation of PPAR
target genes. Indeed, the
dexamethasone-dependent increase in PPAR
expression is associated
with a marked potentiation of the effects of the PPAR
activator
WY-14,643 on the expression of the ACO gene. These experiments provide
evidence that the amount of receptor is a limiting factor and thus that
the regulation of the level of PPAR
expression by glucocorticoids
has impact on the regulation of its target genes. In the whole animal,
the exact physiological conditions in which such a
glucocorticoid-dependent modulation of PPAR
signaling occurs
remain, however, to be investigated. For example, we did not observe
diurnal variations in expression of the ACO gene (data not shown). This
may well be due to the absence of significant levels of endogenous
PPAR
ligand. The same phenomenon was observed in primary
hepatocytes in which the induction of PPAR
expression by
dexamethasone is insufficient to stimulate ACO mRNA levels in the
absence of an activator of the receptor. These observations suggest
that activation of the receptor is a prerequisite for an effect of
glucocorticoids on PPAR
target genes. Investigation of
physiological states in which these conditions are fulfilled, that is
in which PPAR activators/ligand are produced, will possibly give
important clues for the identification of the natural PPAR
ligand.
In conclusion, data in this paper suggest that glucocorticoids have
an important regulatory impact on PPAR expression in
vivo. Physiological situations, such as stress and the diurnal
surge of glucocorticoids, affect PPAR
expression in liver. The
regulation of PPAR
expression provides a control mechanism which,
when coupled to activator availability, regulates the PPAR
action
on its target genes and associated metabolic pathways.