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Defines a Novel Positive Feedback Loop in the Rodent Liver Circadian Clock
Structure and Evolution of Nuclear Receptors (L.C., J.R., V.L.), Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5161, Institut Fédératif de Recherche (IFR) 128 BioSciences Lyon-Gerland, Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure, 69364 Lyon cedex 07, France; Institut National de la Santé et de la Recherche Médicale Unité 371 (O.D.-B.), Laboratoire Cerveau et Vision, IFR 19-Université Claude Bernard Lyon 1, 69675 Bron, France; Université de Nice Sophia Antipolis CNRS Formation de Recherche en Evolution (B.R., F.D.), 06108 Nice cedex 2, France; and Center for Integrative Genomics (N.S.T., L.M., W.W.), University of Lausanne, CH-1015 Lausanne, Switzerland
Address all correspondence and requests for reprints to: Vincent Laudet, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5161, Institut Fédératif de Recherche 128 BioSciences Lyon-Gerland, Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure, 46 allée d’Italie, 69364 Lyon cedex 07, France. E-mail: vincent.laudet{at}ens_lyon.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(PPAR), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPAR influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPAR plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPAR on a potential PPAR response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPAR gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPAR in peripheral clocks. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to the SCN, other peripheral tissues such as liver, heart, kidney (7, 8), as well as isolated cells (9) express clock genes giving rise to circadian rhythms with a different phase from that observed in the SCN. Interestingly, these peripheral clocks can be resetted by alternative routes independently of the SCN, for example by forced change of feeding time (10, 11). Several lines of evidence suggest that the peripheral circadian clocks are not SCN independent but require inputs from the SCN to drive the rhythmicity and ensure an ordered response of the organism to environmental changes (12, 13). Thus, the SCN is believed to coordinate rhythms in the brain and body via a combination of neural and humoral diffusible and synaptic signals (7, 14, 15).
Genetic analyses have identified master clock genes such as clock, bmal1, period (per1, 2), and cryptochrome genes (cry1, 2), as well as the orphan nuclear receptor genes, rev-erb and ror (16, 17). Other transcription factors functioning in the circadian regulation of gene expression, including DBP-related factors (18), Rev-erbß, RORß and -
(19) have also been identified. The clock mechanism mainly involves an integrated network of interacting self-sustained transcriptional-translational feedback loops, composed of positive and negative regulators, which drive their own rhythmic expression and the one of clock-controlled genes to perform a fine tuning of circadian gene expression (20).
Recent reports have highlighted the interplay between circadian oscillators, metabolism, and physiology. Whereas genes involved in the glucose and lipid metabolism are known to exhibit circadian variations (21, 22, 23), molecular studies have revealed a critical role for bmal1 and clock genes in regulating glucose homeostasis (24) and lipid metabolism (25, 26, 27, 28). Moreover, the cross talk between ROR and Rev-erb was shown to be physiologically important for the control of cholesterol and triglyceride metabolism (29, 30, 31). In turn, the peripheral clocks can be coordinately regulated by multiple circulating factors, which are affected by the metabolic status of the organism. Indeed, glucose, one of the major food metabolites that exhibits a plasma diurnal rhythm, is a direct resetting signal in cultured cells by down-regulating per1 and per2 RNA levels (32). The levels of glucose-regulated hormones such as insulin or glucagon immediately up-regulate per1 and per2 expression (13). In addition, other studies have revealed an important role of glucocorticoids and retinoids in the resetting of peripheral clocks (33, 34). Although the elucidation of the mechanisms that govern the connection between metabolism and circadian clock has just begun, it appears that several members of the nuclear receptor family are involved in this pathway.
Evidence has emerged that peroxisome proliferator-activated receptor (PPAR), a member of the nuclear receptor superfamily that regulates the expression of numerous genes involved in lipid metabolism and energy homeostasis, can play a role in the normal circadian regulation. First, PPAR has been identified as a circadian clock-controlled gene with a diurnal rhythm at the mRNA and protein levels in rats and mice in many peripheral organs such as liver, heart, kidney, and to a lesser extent in the SCN, where the central pacemaker is located (7, 35, 36). This circadian expression of PPAR may be in part controlled by hormonal factors, because insulin and glucocorticoids regulate its mRNA expression (35, 37, 38, 39). A recent study has also shown that the circadian expression of PPAR mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner (27). Second, because daily variations in lipogenic and cholesterogenic gene expression are attenuated or abolished in mice in which the PPAR gene has been disrupted, PPAR may be an important mediator for the circadian regulation of lipid metabolism (40, 41). It is now believed that PPAR has a wider general role in transducing hormone messages involved in dietary status (42). These observations thus suggest that PPAR may be required in the control of circadian food-dependent fluctuations in gene expression. Third, PPAR is connected to the regulation of other nuclear hormone receptors such as Rev-erb, because fenofibrate, a PPAR agonist, induces human and rat rev-erb expression in liver through the direct binding of PPAR on an atypical DR2 element located in the rev-erb promoter (30, 43). It was recently shown that CLOCK plays an important role in lipid homeostasis by regulating the circadian transactivation of potential PPAR response element (PPRE)-controlled target genes (26) and of PPAR gene itself via an E-box rich region in vivo and in vitro (27). Fourth, the partner of PPAR, RXR, interacts with CLOCK protein in a ligand-dependent manner and inhibits CLOCK/brain and muscle Arnt-like protein 1 (BMAL1)-dependent activation via an E-box element (34). Other results have also suggested that PPAR deficiency disturbs the normal circadian regulation of certain SREBP-sensitive genes in the liver (40, 44).
Despite much evidence supporting a role of PPAR in metabolic control and energy homeostasis (45) and the accumulation of data connecting metabolism and circadian biology (3, 46, 47), little is known concerning the influence of PPAR on the circadian clock. In the present study, we investigated the mechanisms by which PPAR can influence the pacemakers acting in the SCN and in the liver. We report that PPAR-deficient (PPAR–/–) mice present similar locomotor activity with wild-type (WT) mice without any molecular alteration of clock gene expression in the SCN. Interestingly, we show for the first time that PPAR is a direct regulator of bmal1 expression in liver via its direct binding on a PPRE located on the bmal1 promoter. This regulation is required to maintain the normal circadian oscillation of bmal1 in vivo. Stressing the importance of the regulatory pathway that exists between PPAR and the peripheral clock genes, we show that fenofibrate up-regulates bmal1 gene expression in murine liver and induces circadian rhythm of clock gene expression in cell culture in a PPAR-dependent manner. Reversely, we also observe that BMAL1 is an upstream regulator of the PPAR gene expression. Taken together, our data implicate PPAR in a new regulatory loop that controls peripheral circadian clocks.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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–/– Mice Display Normal Circadian Locomotor Activity and Clock Gene Expression in the SCN on the function of the central circadian oscillator, behavioral analysis of the circadian rhythm was carried out using PPAR–/– and WT littermate mice. Animals were first synchronized for 2 wk to a 12-h light, 12-h dark cycle (12L:12D). Under these light conditions, WT and PPAR–/– mice entrained normally and consolidated their locomotor activity to the dark period of the LD cycle (Fig. 1A
). No difference in the total amount of daily activity was observed between WT and PPAR–/– mice (Fig. 1B). When placed in constant darkness, the PPAR-deficient mice do not display an arrhythmic behavior with endogenous period similar between both genotypes (24.11 ± 0.11 and 24.12 ± 0.08 h, respectively, in PPAR-deficient and WT mice).
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can alter the master oscillator in the SCN at the molecular level, we further compared the circadian expression profiles of clock genes in the SCN of WT and PPAR mutant mice (Fig. 1C
). There is no significant difference in the amplitude and in the phase of the circadian expression of the tested clock genes (bmal1, per2, per3, cry2, and rev-erb). This clearly suggests that there is no major molecular effect of the PPAR deletion at the central clock level and that the expression of PPAR in the SCN is not essential for the basal maintenance of the central circadian timing system.
PPAR Is Required to Maintain the Amplitude of the Circadian Expression of bmal1 in the Murine Liver
To evaluate the role of PPAR on the circadian system of the liver (a peripheral clock), where it is mainly expressed, we analyzed the circadian expression of several clock genes in liver isolated from WT and PPAR mutant mice. As already reported, PPAR expression effectively follows a circadian rhythm in peripheral tissues such as liver, kidney, and muscle (data not shown). Figure 2 shows that all the clock genes tested (bmal1, per1, per2, per3, cry2, and rev-erb) are expressed in a circadian manner with no modification in the phase of their rhythm between both genotypes. By contrast, the amplitudes of bmal1 and per3 expression are drastically affected in PPAR-deficient mice by comparison with the WT, with a significant decrease at circadian time 1 (CT1) and CT21 for bmal1 (where CT0 is subjective day beginning at 0700 h, and CT12 is subjective night beginning at 1900 h) and an increase at CT8 for per3. These data suggest that PPAR does not influence the phase synchronization properties of the liver clock but affects the amplitude of two major clock genes bmal1 and per3.
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Expression and Is Globally Maintained in Liver of PPAR–/– Mice role in the hepatic lipid metabolism during starvation, we hypothesized its potential role in food phase resetting of the liver clock. To examine whether the daily feeding time can affect the phase of the PPAR circadian expression in the liver, WT mice were fed for 2 wk exclusively during the day or during the night. As expected, mice fed exclusively during the day displayed an inversed phase in circadian hepatic expression of bmal1, per1, per3, and rev-erb gene by comparison to mice fed only during the night. Similarly, feeding during the day entirely inversed the phase of liver PPAR expression (Fig. 3A), a result that is in agreement with the demonstration that PPAR is a clock-controlled gene in the liver (7, 36). Control mice fed only during the night displayed a similar phase of hepatic clock gene expression and PPAR than mice fed ad libitum.
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expression in the absence of PPAR (Fig. 3B). Except the bmal1 expression, which was refractory to resetting, per1, per3, and rev-erb genes showed an inversed rhythm of their expression in the liver after daytime-restricted feeding compared with nighttime feeding in the PPAR knockout context. The present results show that feeding time can reset the expression of per1, per3, and rev-erb in the liver of PPAR–/– mice and suggest that bmal1 expression might be controlled by PPAR.
Fenofibrate Induces Expression of Clock Genes in Rat-1 Fibroblasts and Up-Regulates bmal1 Gene Expression in Liver
Because PPAR presents a circadian expression in liver, we first studied whether a shock with a serum-rich medium is able to induce an oscillation of PPAR in the well-established in vitro model Rat-1 fibroblasts (9, 13, 48, 49, 50). As shown in Fig. 4A, PPAR gene expression is induced by a serum shock in Rat-1 fibroblasts with a maximal level reached 12 h after the beginning of the treatment in comparison with the control. The induced-oscillating expressions of rev-erb and cry1 after serum shock were in accordance with previously reported data (9, 51). In addition, rev-erb expression is delayed (peak at 16 h after the serum shock treatment) compared with PPAR expression, which suggests that in Rat-1 fibroblasts PPAR induces rev-erb expression. Similarly to these observations, it seems likely that serum largely participates in the synchronization of the circadian oscillation of PPAR mRNA in fibroblast cultures.
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expression and are classical drugs used in the treatment of dyslipidemias. Using the same in vitro model, we then evaluated whether fenofibrate can induce expression of PPAR gene. The addition of fenofibrate into the serum-free medium triggered a rhythmic expression of PPAR peaking 12–14 h as observed after a serum treatment (Fig. 4B). Fenofibrate induced a PPAR expression 6-fold higher than serum (Fig. 4, A and B).
Fenofibrate can also induce a rhythmic expression of clock genes. The temporal induction by fenofibrate of cry1, rev-erb, and bmal1 expression into the culture medium was almost similar to that observed after a serum-shock: cry1 mRNA level peaked at Zeitgeber time 8 (ZT8) to ZT10 (where ZT0 is time when the light switched on at 0700 h, and ZT12 is time when the light switched off at 1900 h), rev-erb at ZT16–ZT20, and bmal1 at ZT4 (Fig. 4C). Thus, fenofibrate can act as a Zeitgeber in cell culture and trigger a rhythm of clock gene expression.
To determine whether fenofibrate can also reset the liver clock in vivo, the response of bmal1 and rev-erb genes to fenofibrate treatment was compared in liver of WT and PPAR–/– mice (Fig. 4D). In accordance with our previous results, fenofibrate was also able to markedly induce bmal1 mRNA levels in control mice but not in PPAR-deficient mice, suggesting that bmal1 induction by fenofibrate is effectively mediated by PPAR. Similarly, as it was previously reported in rat liver and human hepatocytes (30, 43), administration of fenofibrate in WT mice significantly increased the rev-erb mRNA levels. As expected, rev-erb expression in PPAR knockout mice was not induced by fenofibrate, confirming that murine rev-erb induction by fenofibrate is mediated by PPAR. Taken together, these data strongly suggest that fenofibrate can alter the endogenous rhythm of the liver peripheral clock and in vivo.
PPAR Is a Direct Regulator of bmal1 Expression
To specify the regulation of bmal1 gene expression by PPAR suggested by our analysis of bmal1 expression in the PPAR–/– mice, we analyzed the interaction of PPAR with the bmal1 promoter. First, we performed a bioinformatic research of potential PPAR binding sites (PPREs) on the bmal1 promoter region using the Nubiscan software. Two major PPREs were predicted at the positions –1519 (+, TGGACATGGGTCA) and –4943 (–, AGGGCTGAGGACA), the start site corresponding to the one identified in mouse testis (52). To evaluate whether PPAR binds to the bmal1 gene promoter in vivo, the occupancy of the potential PPRE binding sites by PPAR was analyzed using chromatin immunoprecipitation (ChIP) assays performed on hepatocyte DNA using an anti-PPAR antibody (Fig. 5A). As previously shown in vitro, the DNA encompassing the rev-erb Rev-DR2 site (position –45) was precipitated in vivo by the anti-PPAR antibody after fibrate treatment (Fig. 5A, lower panel, lanes 7 and 9), in accordance with a PPAR-Rev-erb cross talk through competition for binding to the same Rev-DR2 site (43). Moreover, an amplification product was observed when the same DNA samples were PCR-amplified using primers covering the PPRE located at the position –1519 in the bmal1 promoter (Fig. 5A, upper panel, lanes 7 and 9). No amplification product was obtained using primers flanking the site at the position –4943 (middle panel). These data further demonstrate that in mice in vivo, PPAR directly binds to the PPRE site located at the position –1519 of the bmal1 promoter in peripheral oscillators and therefore that bmal1 is a direct PPAR target gene.
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mRNA Expression Is Severely Down-Regulated in the Liver of bmal1–/– Mice expression involves bmal1 in the liver, we analyzed the daily accumulation of PPAR mRNA in the liver of bmal1–/– mice by quantitative PCR. PPAR mRNA expression is rhythmic in bmal1–/– mice with a maximum around ZT12 as observed in WT mice, whereas the amplitude of the peak of expression is significantly dampened (Fig. 5B). This result suggests that BMAL1 is an upstream regulator of PPAR gene and is consistent with the recent observation that PPAR expression is also CLOCK dependent (27). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in Peripheral Clocks gene and its protein into a new positive-regulatory feedback loop in the liver. A model summarizing our main results is shown in Fig. 6. We show that PPAR plays an important role in the endogenous rhythmic property of peripheral clocks in vivo, whereas in the central clock PPAR deficiency does not alter the circadian expression of clock genes. These clock gene expressions are only affected in the liver of PPAR-deficient mice. First, PPAR is not essential to drive the central circadian system because PPAR–/– mice entrained normally without arrhythmic behavior in constant darkness and displayed no alteration in the amplitude and the phase of circadian expression of the clock genes (bmal1, per2, per3, cry2, and rev-erb) compared with WT mice. The lack of PPAR-dependent clock regulation in vivo in the SCN can be due either to the absence of a circadian PPAR function in the central clock or to the presence of another isotype of PPAR (ß, ) or other nuclear receptors that are able to bind to the PPRE site and to exert a compensatory effect. Second, the expression of bmal1, an essential gene of the molecular oscillator, is drastically reduced in the liver of PPAR-deficient mice. This regulation of bmal1 transcription by PPAR is likely through a direct binding of PPAR to the PPRE element located at the position –1519 in the bmal1 promoter. In addition to the established transactivation of the circadian promoter rev-erb by PPAR (43), our data largely support the view that PPAR also associates with the circadian bmal1 promoter in vivo. Interestingly, PPAR expression is strongly decreased in the liver of bmal1-deficient mice compared with WT mice. Because no significant change in hepatic CLOCK level is found in bmal1-deficient mice compared with WT mice (53), this suggests that BMAL1 itself is in return involved in the circadian transactivation of PPAR gene at the level of peripheral oscillators in mice. Other observations have also revealed that CLOCK is involved in the circadian transactivation of PPAR (27) and interacts with its partner RXR (34). We propose that bmal1 is directly and positively regulated by PPAR and that BMAL1 imposes in return a circadian regulation on PPAR transcription.
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Expression Is Resetted by Feeding in Peripheral Clocks is involved in food processing and energy homeostasis (45). A connection between circadian gene regulation, metabolism and energy homeostasis was also established. For example, glucose metabolism (e.g. 6-phosphofructokinase-2, aldolase, and glucose phosphate isomerase) is under the control of the circadian time-keeping system (23, 24). Rhythmic expression of numerous enzymes and transcription factors involved in protein and amino acid metabolism (e.g. serine dehydratase, DBP, and 3-hydroxy-3-methylglutaryl coenzyme A reductase) (7, 54), in fat metabolism (e.g. cholesterol 7-hydroxylase, PPAR, HMGCoA lyase and reductase) (7, 35, 55), or in detoxification process [e.g. steroid 15-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5), Cyp2e1, Cyp17, and glutathione-S-transferase 
2] (23, 56) was also observed in liver. Herein, we show that the circadian expression of PPAR is resetted by feeding time and that, in the absence of PPAR, clock gene expression in liver is entrained by reversed feeding time, suggesting that PPAR does not play a major role in food resetting. We underline that, after an extended duration of daytime feeding, the food-imposed reversed phase of circadian gene expression in peripheral liver is similar between WT and PPAR-deficient mice. This is probably due to PPAR-independent signaling pathway that plays a major role in the phase resetting of circadian gene expression by feeding time.
Fibrates as a Resetting Signal in Cell Cultures and Peripheral Clocks
Previous studies have shown that PPAR expression is positively controlled by glucocorticoids and fibrates and negatively by insulin (37, 38, 39). Fibrates are also known to up-regulate the expression of rev-erb in the rat liver and in both rat and human primary hepatocyte cultures (30, 43). In vitro, PPAR mRNA was induced in rat fibroblasts culture after both a serum shock and, more interestingly, a fenofibrate treatment. Moreover, fenofibrate efficiently stimulates the rhythmic expression of several clock genes such as cry1, bmal1, and rev-erb. This suggests that fenofibrate is able to entrain rhythmic PPAR and clock gene expressions in Rat-1 fibroblasts. In vivo, rev-erb mRNA level is up-regulated by fenofibrate in the mouse liver of WT animals. Because this up-regulation of rev-erb gene by fibrates is not observed in the PPAR-deficient mice, we confirm in vivo that the induction of rev-erb gene expression by fibrates is mediated by PPAR at the transcriptional level. Interestingly, the increased accumulation of PPAR after fibrate treatment leads in turn to a higher level of expression of bmal1 in WT mice. Other convincing evidence that the fibrate effect on the bmal1 expression is exerted at the transcriptional level via the PPAR protein is that fibrates have no effect on the bmal1 expression in the PPAR–/– mice. Taken together, these results suggest that PPAR could play a role in integrating chemical signals inside the liver.
How Circadian Rhythm Might Influence a Fibrate Therapy?
To date, fibrates are clinically used as hypolipidemic drugs that lower plasma cholesterol and triglycerides. They exert their effect by regulating the expression of several key genes implicated in lipid metabolism via PPAR activation. Interestingly, fibrate therapy represents a cost-effective approach in the clinical management and the prevention of cardiovascular diseases in a growing population suffering from lifestyle-induced metabolic dysfunctions such as obesity, insulin resistance, and diabetes (57). Although important differences in lipid metabolism exist between mice and human, including the function of PPAR (58, 59), our in vitro and in vivo studies suggest that the regulation by fibrates of the circadian expression of clock genes may influence the success of a treatment because it would suggest a potential induction and subsequently a dysfunction of their expression after fibrate supply in patients. The validity of this hypothesis remains to be demonstrated in humans. Therefore, one additional question to address is how fibrate administration at a selected time of the day can impact the efficacy and the success of the treatment. It will be of interest to decipher the molecular mechanisms involved in the circadian expression of clock genes and PPAR-regulated genes in presence or absence of fibrates to provide new insight in the downstream circadian physiological and cellular processes governed by PPAR itself. Similarly, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of dyslipidaemias.
In summary, our data indicate that PPAR is a specific element of the liver oscillatory clock in mammals and plays an important role in integrating signals into the clock machinery. We clearly demonstrate that, in vivo, PPAR is required to maintain normal circadian oscillation of the master clock gene bmal1 in liver. This regulation occurs via a direct binding of PPAR on a PPRE located in the bmal1 promoter. In addition, BMAL1 is an upstream regulator of the PPAR expression. This finding provides a new regulatory pathway for the circadian system and suggests that some transcription factors may have a specific role in the peripheral clocks. Further studies are now required to determine the exact impact of circadian rhythms on the metabolic processes governed by PPAR in peripheral organs. This can be addressed by investigating the circadian regulation of PPAR target genes and some of the downstream targets in the peripheral clock in WT, PPAR–/–, and other clock mutant mice. The complete elucidation of the signaling elements involved in the interactions between central and peripheral clocks and the mechanisms that govern the interplay between metabolism and circadian oscillators will also have a major impact on the circadian field in the next future.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animal Experiments
Purebred WT and homozygous PPAR–/– mice on an SV129 background were used. All experiments were done with male mice between 6 and 8 wk of age. Animals were kept under a 12L:12D cycle, and food and drinking water were available ad libitum, except when indicated otherwise. The experimental protocols of the current research were approved by the rules and regulations of French veterinary services.
Locomotor Activity Recording
Adult male mice (n = 8 for both WT and PPAR–/–) were exposed to 12L:12D cycle for at least 2 wk. For monitoring locomotor activity, mice were housed individually in cages equipped with infrared motion captors placed over the cages and a computerized data acquisition system (Circadian Activity Monitoring System, Institut National de la Santé et de la Recherche Médicale, France). Activity records were analyzed with the Clocklab software package (Actimetrics, Evanston, IL). For each animal, the total duration of activity was determined every 2 or 12 h during the LD cycle and then averaged for WT and knockout mice. Animals were then allowed to free run in constant darkness (DD) for at least 15 d. The endogenous period in DD was subsequently determined using the Clocklab software.
Circadian Expression of Clock Genes in Wild-Type vs. PPAR–/– Mice
Twelve WT and 12 PPAR–/– mice were maintained for a period of 2 wk in a 12L:12D cycle and transferred in DD the day before the kill. Livers and SCN were removed at CT1, CT8, CT14, and CT21; stored at –70 C until RNA extraction; and analyzed by quantitative RT-PCR. The experiment was done twice.
Restricted Feeding
Mice (n = 24 both for WT and PPAR–/–) fed during the day received food when light was on (0700 to 1900 h), whereas mice (n = 24 both for WT and PPAR–/–) fed during the night received food from 1900 to 0700 h for 2 wk. Water was freely available over the experimental period. As controls, WT and PPAR–/– mice (n = 24 for both) were fed ad libitum. Mice were transferred in DD the day before killing. Livers were dissected at indicated circadian times (CT1, CT8, CT14, CT21), stored at –70 C until RNA extraction, and analyzed by quantitative-RT-PCR.
Fenofibrate Response
In this experiment, mice (n = 32 both for WT and PPAR–/–) were treated for 2 wk with fenofibric acid (vehicle DMSO) mixed in the drinking water at the final concentration of 7 mM. Control animals (n = 32 both for WT and PPAR–/–) were treated with vehicle in the drinking water. Livers were removed at CT1, CT8, CT14, and CT21, stored at –70 C until RNA extraction, and analyzed by quantitative RT-PCR.
Analysis of bmal1–/– Mice
WT and homozygous bmal1–/– mice on B6 background (both females and males aged from 8–14 wk provided by C. Bradfield, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI) were kept under 12L:12D cycles and fed ad libitum. Livers were removed at indicated Zeitgeber time. Reverse-transcribed total RNAs from three to five animals per time point were analyzed by quantitative RT-PCR as described below.
RNA Extraction and Quantitative RT-PCR
Total RNAs were prepared from cells or from organs according to the manufacturer’s instructions (Sigma) and reverse transcribed using random primers and MMLV Reverse Transcriptase (Invitrogen, San Diego, CA). cDNA were then used as template for a quantitative real-time PCR assay using the QuantiTect SYBR Green PCR reagents (Qiagen, Valencia, CA) and the DNA Engine Opticon system (MJ Research, Cambridge, MA). Each couple of oligonucleotides used were designed to hybridize on different exons. The sequences of forward and reverse primers were as follows: bmal1, forward, 5'-CCAAGAAAGTATGGACACAGACAAA-3'; bmal1, reverse, 5'-GCATTCTTGATCCTTCCTTGGT-3'; cry1, forward, 5'-CTGGCGTGGAAGTCATCGT-3'; cry1, reverse, 5'-CTGTCCGCCATTGAGTTCTATG-3'; cry2, forward, 5'-TGTCCCTTCCTGTGTGGAAGA-3'; cry2, reverse, 5'-GCTCCCAGCTTGGCTTGAA-3'; per1, forward, 5'-GGAGACCACTGAGAGCAGCAAG-3'; per1, reverse, 5'-CGCACTCAGGAGGCTGTAGGC-3'; per2, forward, 5'-ATGCTCGCCATCCACAAGA-3'; per2, reverse, 5'-GCGGAATCGAATGGGAGAAT-3'; per3, forward, 5'-GGCGTTCTACGCGCACACTGC-3'; per3, reverse, 5'-CGCTGGTGCACATTCATACTGCG-3'; ppar, forward, 5'-CGCTATGAAGTTCAATGCCTT-3'; ppar, reverse, 5'-TGCAACTTCTCAATGTAGCC-3'; rev-erb, forward, 5'-CATGGTGCTACTGTGTAAGGTGTGT-3'; rev-erb, reverse, 5'-CACAGGCGTGCACTCCATAG-3'; 36B4, forward, 5'-ACCTCCTTCTTCCAGGCTTT-3'; and 36B4, reverse, 5'-CCCACCTTGTCTCCAGTCTTT-3'.
The efficiency (>95%) and the specificity of the amplification were controlled by generating standard curves and carrying out melting curves and agarose gels of the amplicons, respectively. The relative levels of each RNA were calculated by 2–CT (CT standing as the cycle number in which SYBR Green fluorescence exceeds a constant threshold value) and normalized to the corresponding noncyclic 36B4 RNA levels. The presented values are means ± SEM of duplicates of the same reaction for at least three different mice or three experimental points.
The significance of differences was assessed by distribution-free two-way ANOVA. Paired Student’s tests were used to compare WT and PPAR–/– or bmal1–/– mice. Results are presented as mean ± SEM. Differences were considered significant when P < 0.05.
ChIP Assays
ChIP experiments were performed as already described in Ijpenberg et al. (60). Briefly, WT and PPAR–/– mice (n = 3) were fed for 5 d with either Wy14,643 (50 mg/kg·d) or vehicle. Immunoprecipitation of liver extracts was done using a PPAR antibody and the immunoprecipitated DNA was PCR amplified using primers flanking either the rev-erb Rev-DR2 (GTGTCACTGGGGC) or potential PPRE (usually AGGTCANAGGTCA) on the bmal1 promoter predicted using the computer program NUBISCAN available at the following website: www.nubiscan.unibas.ch (61). An equal volume of nonprecipitated genomic DNA (Input) was amplified as positive control. One fifth of PCR products were separated on an ethidium bromide-stained 2% agarose gel.
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ACKNOWLEDGMENTS |
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–/– mice. |
FOOTNOTES |
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Present address for L.C.: ANIMAGE, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5515, 59 Boulevard Pinel, 69677 Bron cedex, France.
L.C., J.R., O.D.-B., B.R., N.S.T., L.M., F.D., W.W., and V.L. have nothing to declare.
First Published Online March 23, 2006
Abbreviations: BMAL1, Brain and muscle Arnt-like protein 1; ChIP, chromatin immunoprecipitation; CT, circadian time; DD, constant darkness; LD, light-dark; 12L:12D, 12-h light, 12-h dark; PPAR, peroxisome proliferator-activated receptor ; PPRE, peroxisome proliferator response element; PSG, penicillin/streptomycin/glutamine; SCN, suprachiasmatic nucleus; WT, wild type; ZT, Zeitgeber time.
Received for publication January 30, 2006. Accepted for publication March 15, 2006.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(PPAR) in mice. Biochem J 386:575–581[CrossRef][Medline]. J Biol Chem 276:2865–2871 and Rev-erb mediate the species-specific regulation of apolipoprotein A-I expression by fibrates. J Biol Chem 273:25713–25720. J Biol Chem 272:22401–22404 gene is stimulated by stress and follows a diurnal rhythm. J Biol Chem 271:1764–1769 gene by glucocorticoids. J Biol Chem 269:24527–24530-deficient mice. J Lipid Res 42:328–337 (PPAR-). Adv Enzyme Regul 42:227–247[CrossRef][Medline] expression in liver via a novel peroxisome proliferator-activated receptor response element. Mol Endocrinol 13:400–409 in the control of SREBP activity and lipid synthesis in the liver. Biochem J 389:413–421[CrossRef][Medline] gene is a target of the circadian clock pacemaker. J Mol Endocrinol 33:585–608-dependent mechanism. Biochim Biophys Acta 1581:89–99[Medline] hydroxylase gene may involve the liver-enriched bZIP protein DBP. Genes Dev 7:1871–1884-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol Cell Biol 19:6488–6499 expression in human liver. Mol Pharmacol 53:14–22 to guinea pig hepatocytes confers increased responsiveness to peroxisome proliferators. Cancer Res 59:4776–4780NURSA Molecule Pages Link:
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) and PPAR
). The distribution of activity was determined every 12 h during LD cycle. Shadowed areas indicate the dark period. Results are expressed as the means ± SEM of values from eight animals per group. C, Circadian expression of bmal1, per2, per3, cry2, and rev-erb
) and PPAR
). Real-time PCR was used to determine transcript levels at four circadian times (CT1, CT8, CT13, CT21). Transcript levels are displayed as relative quantity (RQ) after normalization to the noncyclic 36B4 expression levels in the same sample. Results are expressed as the means ± SEM of values of two independent experiments, each realized with three animals for both genotypes at each time point. Statistically significant differences between WT and PPAR
), and rev-erb
