©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Peroxisome Proliferator-activated Receptor Gene Is Stimulated by Stress and Follows a Diurnal Rhythm (*)

(Received for publication, October 12, 1995)

Thomas Lemberger (1)(§) Régis Saladin(§) (2) Manuel Vázquez (1)(¶) Françoise Assimacopoulos (3) Bart Staels (2)(**) Béatrice Desvergne (1) Walter Wahli (1)(§§) Johan Auwerx (2)(¶¶)(§§)

From the  (1)Institut de Biologie Animale, Université de Lausanne, CH-1015 Lausanne, Switzerland, the (2)Laboratoire de Biologie des Régulations chez les Eucaryotes, U.325 INSERM, Département d'Athérosclérose, Institut Pasteur, 59019 Lille, France, the (3)Département de Biochimie Médicale, Centre Médical Universitaire, CH-1211 Geneva, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPARalpha 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 PPARalpha 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 PPARalpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPARalpha 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 PPARalpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPARalpha expression on PPARalpha target gene activity, high glucocorticoid-dependent PPARalpha 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 PPARalpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPARalpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.


INTRODUCTION

The peroxisome proliferator-activated receptors (PPARs) (^1)are orphan nuclear hormone receptors(1, 2) . To date, three different subtypes, PPARalpha, PPARbeta 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 PPARalpha 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 beta-oxidation pathway(15) . Definitive proof that PPARalpha is the major mediator of these effects was provided by the absence of the typical hepatic response to peroxisome proliferators in PPARalpha-deficient mice generated by targeted disruption of the PPARalpha 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 PPARalpha target genes were identified that contain one or several PPAR-responsive elements in their promoter(1, 2) . To date, the known PPARalpha 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 beta-oxidation (acyl-CoA oxidase (ACO), Refs. 3, 18, and 19, and bifunctional enzyme, (20) ), (iii) mitochondrial beta-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 PPARalpha, 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 PPARalpha, 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 PPARalpha suggests that fatty acids could represent biological activators(5, 18) . According to this hypothesis, PPARalpha could function as a fatty acid sensor, which would allow the fatty acids to regulate their own metabolism(1, 2) .

PPARalpha mRNA is predominantly expressed in tissues capable of oxidizing fatty acids, such as brown adipose tissue, liver, heart, kidney, and muscle(8) . Absence of PPARalpha 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 PPARalpha is important for the proper regulation of these genes in vivo(16) . In fact, PPARalpha expression in adult rat liver is subject to marked interindividual variations for so far unknown reasons. (^2)We and others (28, 29) have shown recently that PPARalpha 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 PPARalpha 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 PPARalpha mRNA levels increase in rat liver during an immobilization stress situation and follow a diurnal rhythm. The impact of variations in the levels of PPARalpha expression on the regulation of a prototypical PPARalpha target gene, the ACO gene, is furthermore analyzed using hepatocytes in primary culture.


MATERIALS AND METHODS

Animals and Treatment

Eight-week-old male Fisher 344 rats (BRL, Basel, Switzerland) were group-housed and had free access to water and food. The animals were kept on a 12-h light-dark cycle (light from 8:00 a.m. to 8:00 p.m. in the stress and dexamethasone experiments; light from 7:30 a.m. to 7:30 p.m. in the diurnal variation experiment). All animals were accustomed to this cycle during at least 14 days. Rats were stressed by immobilization in transparent plastic tubes (5 cm diameter, 20 cm long) with small holes in the front allowing breathing and a hole at the back for the tail. Immobilization started at 9:00 a.m. The unstressed control animals were not manipulated during the duration of the experiment. Stressed (n = 3) and control (n = 3) rats were sacrificed at the same time. In the RU 486 experiments, both the stressed (n = 3) and control (n = 3) animals were injected subcutaneously with either vehicle or 30 mg/kg body weight (BW) of RU 486 dissolved in 200 µl of propylene glycol. In the dexamethasone treatment experiments, animals were injected intraperitoneally, under mild Forene (Abbott, Cham, Switzerland) anesthesia, with either vehicle (n = 3) or 40 µg/kg BW of dexamethasone dissolved in 500 µl of saline containing 1% ethanol (n = 3). In the diurnal variation experiment, the rats (12 animals) were sacrificed at precise time points (9:00 a.m., 3:30 p.m., 5:00 p.m., and 6:30 p.m.). For each time point, the animals (n = 3) were sacrificed on three consecutive days. Since, in this experiment, the rats were housed in groups of five animals in four independent cages, the last animals killed were never alone, thus preventing an isolation stress. Furthermore, the rats were picked randomly among the four cages. The rats used in the dexamethasone and diurnal expression experiments were sacrificed by decapitation under Forene anesthesia, whereas the rats in the stress experiments were sacrificed by exsanguination under ether anesthesia. Blood samples were collected in heparinized tubes (Milian, Geneva) and centrifuged. Plasma was frozen until analyzed. The tissues were rapidly dissected and frozen in liquid nitrogen.

Corticosterone Measurements

Plasma corticosterone levels were measured by radioimmunoassay(30) .

RNA Preparation and Analysis

Total RNA was prepared using either the acid guanidinium thiocyanate-phenol-chloroform extraction method (31) or using the TRIzol Reagent (Life Technologies, Inc.). Quantification of PPARalpha and L27 mRNA levels by RNase protection assay were performed as described(28) . ACO mRNA was detected by RNase protection using a rat ACO probe corresponding to the 447 nucleotide long SalI-SacI fragment of the full-length cDNA(32) .

Antibodies

A polyclonal antibody raised against the AB domain of mPPARalpha was generated as follows. A cDNA fragment corresponding to the 101 first amino acids of mPPARalpha was amplified by PCR from the full-length cDNA ((4) , upstream primer: 5`-CCGGATCCATGGTGGACACAGAGAGCC-3`, downstream primer: 5`-GCGCCCGGGATGTTCAGGGCACTGCCGG-3`) and digested with BamHI and SmaI. This fragment was cloned into the bacterial expression pQE-9 vector (Qiagen) using the BamHI and Klenow-filled HindIII sites. The same fragment was inserted into the pGEX-2T vector (Pharmacia Biotech Inc.) using the BamHI and SmaI sites. The pQE-9 construct was used to overexpress the mPPARalpha/AB domain fused to a 6 times His tag in XL-1 bacteria (Stratagene). The resulting soluble polypeptide was purified from bacterial extract by affinity chromatography on a nickel-nitrilotriacetic acid-agarose column under native conditions according to the manufacturer's instructions (Qiagen, Hilden, Germany). The purified polypeptide was injected subcutaneously into KOBU rabbits (one primary and four booster injections of 200 µg). Serum was collected 10 days after the final antigen injection. To affinity purify the serum, an antigen-coupled column was prepared. The pGEX-2T construct described above was used to overexpress a GST-PPARalpha/AB domain fusion protein, which was purified onto a glutathione-Sepharose column (Pharmacia) and coupled to a N-hydroxysuccinylimide-activated HiTrap column (Pharmacia). The resulting affinity column was then used to purify the immune serum. The resulting polyclonal antibody cross-reacts with rat PPARalpha, which is 98% conserved at the amino acid level in the AB region. Preimmune serum IgGs were purified using a protein G-Sepharose 4 fast flow column (Pharmacia).

Nuclear Extracts and Western Blotting

Nuclei were prepared as follows. Liver samples (0.5 g) or cultured hepatocytes (2.4 times 10^7 cells) were homogenized in 0.5 M sucrose, 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 25 mM KCl (0.5 M sucrose TEKS). Cells were lysed with 0.5% Triton X-100 for 30 min at 4 °C. The homogenate was then layered on a 0.9 M sucrose TEKS cushion and centrifuged at 2000 times g for 20 min. Nuclei were resuspended in 40% glycerol, 50 mM Tris-Cl, pH 8, 5 mM MgCl(2), 0.1 mM EDTA and stored at -70 °C. The concentrations of the nuclei were determined by measuring A at 260 nm in 5 M NaCl. Nuclei were lysed directly in SDS-gel loading buffer and loaded onto a 10% polyacrylamide-SDS gel. After electrotransfer on nitrocellulose, equal loading was checked by staining the blots with 0.2% Ponceau S red. The blots were blocked 1 h at 25 °C with 5% non-fat dry milk in 25 mM Tris-Cl, pH 8.3, 140 mM NaCl, 2 mM KCl, and 0.05% Tween 20 (NFDM TBS-Tween) and incubated overnight at 4 °C with the primary antibody at a dilution of 1:1000 in 5% NFDM TBS-Tween. Six 10-min washes at 25 °C with 5% NFDM TBS-Tween were performed. The filters were subsequently incubated 2 h at 25 °C with the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel, Turnhaut, Belgium), at a dilution of 1:1000 in 5% NFDM TBS-Tween and washed six times 10 min in TBS-Tween. Signal detection was achieved by chemiluminescence with the ECL system (Amersham) and 15-s to 5-min exposure to x-ray films. Signals were quantified using an Elscript 400-AT/SM densitometer (ATH, Neuried, Switzerland).

Hepatocytes Primary Cultures

Rat hepatocytes were isolated by collagenase perfusion (33) of livers from 200-250 g rats (cell viability higher than 85% by trypan blue exclusion test). The hepatocytes were cultured in monolayer (1.5 times 10^5 cells/cm^2) in Williams' E medium (Life Technologies, Inc.) supplemented with 5% fetal calf serum and antibiotics, at 37 °C in a humidified atmosphere of 5% CO(2)/95% air. Treatments with WY-14,643 (100 µM in ethanol) and dexamethasone (1 µM in ethanol) were started immediately after seeding.


RESULTS

Stress Induces PPARalpha mRNA Expression

Physical and psychological stress triggers a multihormonal response that mainly comprises the release of catecholamines by the sympathetic nervous system and glucocorticoids by the adrenal cortex(34, 35) . The glucocorticoid levels in blood are elevated during experimental stress situations such as swimming, heat or cold exposure, photic or acoustic stimuli, and forced immobilization(36, 37, 38) . Since the PPARalpha gene is under direct control of glucocorticoid hormones in rat hepatocytes cultured in vitro(28, 29) , we used stress as an in vivo paradigm to study the regulatory effects of circulating glucocorticoids on the expression of the PPARalpha gene. In this study, an immobilization protocol was used because it is not associated with an increase of physical activity, in contrast to swimming for example. The reason to avoid experimental protocols requiring physical activity was that PPARs are involved in energy homeostasis(1) . PPARalpha expression was analyzed in rats stressed by forced immobilization during 4 h. All stress experiments were started at 9 a.m. to circumvent interference with the diurnal variations of plasma corticosterone levels (see below). For the same reason, unstressed control animals were sacrificed at the same time as the stressed animals. The 4-h immobilization led to a 3-fold increase in the plasma levels of corticosterone, which is the major active glucocorticoid in rats (Fig. 1C). Total RNA was extracted from liver and hippocampus, a structure in the central nervous system that contains significant amounts of PPARalpha (39) and that has been described as one of the regions of the brain most sensitive to stress and glucocorticoids(40) . Rat PPARalpha mRNA levels, as well as the levels of the mRNA of the large ribosomal subunit 27-kDa protein (L27) as a control, were assayed by RNase protection. After a 4-h immobilization, stressed animals displayed a 4.5-fold increase in the PPARalpha mRNA levels in liver relative to the unstressed animals (Fig. 1, A and B). Stress is therefore a potent physiological inducer of PPARalpha expression in liver. In contrast to the liver, no significant variation of PPARalpha mRNA levels could be detected in the hippocampus (Fig. 1, A and B).


Figure 1: Stress stimulates PPARalpha 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 PPARalpha 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 PPARalpha mRNA than the unstressed control animals (C, n = 3). In contrast, there is no variation in PPARalpha mRNA levels in the hippocampus of the same animals (right panel). B, graphic representation of PPARalpha mRNA levels in liver and hippocampus of the control (C) and stressed (S) animals. PPARalpha mRNA levels were normalized to those of L27. The PPARalpha 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 PPARalpha 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 PPARalpha mRNA levels was analyzed. In the saline-injected animals, the 4-h immobilization produced a significant 3-fold increase of PPARalpha mRNA levels in liver (Fig. 2). In marked contrast, the stress-dependent induction of PPARalpha 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 PPARalpha expression by stress.


Figure 2: RU 486 inhibits the stress-dependent stimulation of PPARalpha 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 PPARalpha 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 PPARalpha mRNA expression induced by stress, the acute injection of exogenous glucocorticoids should lead to increased PPARalpha 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 PPARalpha 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 PPARalpha 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 PPARalpha 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.



Cycling of PPARalpha Expression

In rats, similar to the situation in other mammals, the circulating levels of glucocorticoids are subject to diurnal variations. The plasma levels of corticosterone, which are low in the morning, increase in the afternoon to reach a maximum about 2-3 h before the light-dark switch(41) . In view of the results presented above, we expected the levels of PPARalpha mRNA to follow a similar diurnal rhythm. Thus, we compared PPARalpha mRNA expression in liver in the morning to its expression in the afternoon. The animals were kept on a 12-h light-dark cycle, with the light-dark switch at 7:30 p.m. Under these conditions, the peak of circulating corticosterone is expected to occur approximately at 5:00 p.m., which was indeed observed (Fig. 4A) Hence, liver samples were taken from animals sacrificed in the morning (9:30 a.m.) and at three different time points in the afternoon (3:30 p.m., 5:00 p.m., and 6:30 p.m.). The analysis was performed over three consecutive days to assess the periodic nature of the variations of PPARalpha expression and to exclude the possibility of an isolated stress having affected the animals. The mean values for PPARalpha mRNA levels determined for each of the time point are higher in the afternoon than at 9:30 a.m. (3.5-fold at 3:30 p.m.; 4-fold at 5:00 p.m.; 3-fold at 6:30 p.m., Fig. 4B). These variations of PPARalpha mRNA levels correlate well with the diurnal variations of plasma corticosterone levels (Fig. 4A), which strongly suggests that the PPARalpha gene responds to the diurnal variations of circulating corticosterone.


Figure 4: Diurnal variations of PPARalpha 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 PPARalpha mRNA levels at each time point (n = 3), determined as described in the legend to Fig. 1. C, PPARalpha protein levels in liver nuclear extracts as analyzed by Western blotting. The affinity-purified anti-PPARalpha antibody detects a protein of about 55 kDa corresponding to the PPARalpha 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-PPARalpha/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 PPARalpha mRNA levels (solid line, box) and protein levels (dashed line, ) are plotted successively in the sequence the animals were killed during the 3 days of the experiment. PPARalpha 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 PPARalpha mRNA levels resulted into changes in PPARalpha protein expression, the relative levels of the receptor were measured in liver nuclear extracts. PPARalpha protein was detected on Western blots using an anti-PPARalpha antibody. This antibody detects a major band at 55 kDa, which corresponds to the predicted size of PPARalpha. 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, PPARalpha 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 PPARalpha 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).

Dexamethasone Potentiates the Induction of the ACO Gene by WY-14,643

Animal treatments by peroxisome proliferators, such as WY-14,643, produce multiple effects. Since specific PPAR antagonists have not yet been identified, it is difficult to distinguish between the PPAR-mediated direct effects of these hypolipidemic drugs and indirect effects involving metabolic or hormonal feedback mechanisms. Thus, we used hepatocytes in primary culture as an in vitro model to test whether the amount of PPARalpha protein is a limiting factor for the induction of its target genes. Dexamethasone provokes a 4-fold increase of PPARalpha protein level after 6 h of treatment (Fig. 5A). A similar result was obtained after 24 h of treatment (data not shown). It was thus possible to test if an increase of the amount of PPARalpha protein could potentiate the stimulation of the ACO gene expression by WY-14,643, a well characterized peroxisome proliferator and activator of PPARalpha(8) . The ACO gene, which encodes the rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, is controlled by PPARalpha through a specific response element(3) . Hepatocytes cultured in the presence or absence of dexamethasone (1 µM) were treated during 24 h with WY-14,643 (100 µM) or vehicle. As expected, addition of dexamethasone efficiently stimulated the expression of PPARalpha mRNA (6-fold, Fig. 5B, lanes 3 and 4). However, dexamethasone alone had no effect on ACO gene expression, presumably because of a lack of PPAR activators (Fig. 5B, lane 3, and C). In contrast, when hepatocytes were treated with WY-14,643 alone, a 3-fold increase of ACO mRNA levels was observed (Fig. 5B, lane 2, and C). Furthermore, simultaneous treatment of the hepatocytes with both WY-14,643 and dexamethasone increased the ACO mRNA levels 6.5-fold (Fig. 5B, lane 4, and C). Thus, dexamethasone provokes a marked potentiation of the induction of ACO gene by WY-14,643. These results strongly suggest that the amount of receptor is a limiting factor for the stimulation of PPARalpha target genes.


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 PPARalpha 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, PPARalpha, 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.




DISCUSSION

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 PPARalpha 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 PPARalpha gene expression in liver during stress situations. Interestingly, stress was unable to modify PPARalpha 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 PPARalpha gene promoter. Alternatively, brain-specific factors might inhibit this regulation. In liver, the induction of PPARalpha mRNA is a fast response since it can be observed already 4 h after immobilization or agonist injection. The rapid regulation of PPARalpha mRNA expression in vivo reported herein argues for a similar direct transcriptional effect of glucocorticoids on PPARalpha 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 beta-oxidation pathway and ketogenesis is stimulated(42) . Remarkably, PPARalpha regulates genes involved in the activation of fatty acids as well as in the beta-oxidation and ketogenesis pathways(1) . Thus, stimulation of PPARalpha 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 PPARalpha 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 PPARalpha gene to the levels of circulating corticosterone. The diurnal variations of PPARalpha mRNA is closely followed by a parallel cycling of PPARalpha protein suggesting that PPARalpha mRNA is efficiently translated. Furthermore, the cyling of the PPARalpha 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 PPARalpha 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 PPARalpha 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 PPARalpha protein levels.

The investigation of the glucocorticoid-dependent regulation of PPARalpha 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 PPARalpha-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 PPARalpha and activation of the receptor are necessary for a maximal stimulation of PPARalpha target genes. Indeed, the dexamethasone-dependent increase in PPARalpha expression is associated with a marked potentiation of the effects of the PPARalpha 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 PPARalpha 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 PPARalpha 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 PPARalpha ligand. The same phenomenon was observed in primary hepatocytes in which the induction of PPARalpha 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 PPARalpha 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 PPARalpha ligand.

In conclusion, data in this paper suggest that glucocorticoids have an important regulatory impact on PPARalpha expression in vivo. Physiological situations, such as stress and the diurnal surge of glucocorticoids, affect PPARalpha expression in liver. The regulation of PPARalpha expression provides a control mechanism which, when coupled to activator availability, regulates the PPARalpha action on its target genes and associated metabolic pathways.


FOOTNOTES

*
This work was supported by the Etat de Vaud, the Swiss National Science Foundation, CNRS, INSERM, and by grants from the Fondation pour la Recherche Médicale (FRM) and the Association pour la Recherche sur le Cancer (ARC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors have contributed equally to this work.

Recipient of an European Science Foundation grant.

**
Chargé de Recherche.

§§
Correspondence should be addressed either to W. Wahli: Institut de Biologie Animale, Bâtiment de Biologie, Université de Lausanne, CH-1015 Lausanne, Switzerland. Tel.: 41-21-692-4110; Fax: 41-21-692-4105; walter.wahli{at}iba.unil.chunil.ch or to J. Auwerx, LBRE U325 INSERM, Institut Pasteur, 1 Rue Calmette, 59019 Lille Cédex, France. Tel.: 33-20-877752; Fax: 33-20-877360.

¶¶
Directeur de Recherche of the CNRS.

(^1)
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; ACO, acyl-CoA oxidase; BW, body weight.

(^2)
T. Lemberger, W. Wahli, and J. Auwerx, unpublished results.


ACKNOWLEDGEMENTS

We thank Roussel Uclaf, Romainville, France, for the gift of RU 486.


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