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Endocrinology, doi:10.1210/en.2008-0285
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Endocrinology Vol. 149, No. 10 5262-5271
Copyright © 2008 by The Endocrine Society

Bezafibrate, a Peroxisome Proliferator-Activated Receptors Agonist, Decreases Body Temperature and Enhances Electroencephalogram Delta-Oscillation during Sleep in Mice

Sachiko Chikahisa, Kumiko Tominaga, Tomoko Kawai, Kazuyoshi Kitaoka, Katsutaka Oishi, Norio Ishida, Kazuhito Rokutan and Hiroyoshi Séi

Departments of Integrative Physiology (S.C., H.S.), of Stress Science (K.T., T.K., K.R.), and of Physiology (K.K.), Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan; Clock Cell Biology Research Group (K.O., N.I.), Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan; and Graduate School of Life and Environmental Sciences (N.I.), University of Tsukuba, Tsukuba, Ibaraki 305-8502, Japan

Address all correspondence and requests for reprints to: Hiroyoshi Séi, M.D., Department of Integrative Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan. E-mail: sei{at}basic.med.tokushima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor family. PPARs play a critical role in lipid and glucose metabolism. We examined whether chronic treatment with bezafibrate, a PPAR agonist, would alter sleep and body temperature (BT). Mice fed with a control diet were monitored for BT, electroencephalogram (EEG), and electromyogram for 48 h under light-dark conditions. After obtaining the baseline recording, the mice were provided with bezafibrate-supplemented food for 2 wk, after which the same recordings were performed. Two-week feeding of bezafibrate decreased BT, especially during the latter half of the dark period. BT rhythm and sleep/wake rhythm were phase advanced about 2–3 h by bezafibrate treatment. Bezafibrate treatment also increased the EEG delta-power in nonrapid eye movement sleep compared with the control diet attenuating its daily amplitude. Furthermore, bezafibrate-treated mice showed no rebound of EEG delta-power in nonrapid eye movement sleep after 6 h sleep deprivation, whereas values in control mice largely increased relative to baseline. DNA microarray, and real-time RT-PCR analysis showed that bezafibrate treatment increased levels of Neuropeptide Y mRNA in the hypothalamus at both Zeitgeber time (ZT) 10 and ZT22, and decreased proopiomelanocortin-{alpha} mRNA in the hypothalamus at ZT10. These findings demonstrate that PPARs participate in the control of both BT and sleep regulation, which accompanied changes in gene expression in the hypothalamus. Activation of PPARs may enhance deep sleep and improve resistance to sleep loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor family. PPARs, including three known isotypes, PPAR{alpha}, PPAR{gamma}, and PPARβ/{delta}, play a critical physiological role in regulating energy homeostasis, lipid metabolism, and inflammation (1, 2, 3). Like other nuclear hormone receptors, each PPAR forms a heterodimer with a retinoid X receptor (RXR) and binds to its targeted response element in the promoter (1, 2, 4).

PPAR{alpha} is expressed in the liver and other metabolically active tissues, including striated muscle, kidney, and pancreas (1, 5, 6). PPAR{alpha} is known for its role in inducing transcription of enzymes and transporters required for fatty acid oxidation and ketogenesis in response to fasting (1, 2, 3, 7, 8, 9, 10). The activation of PPAR{alpha} stimulates target gene transcription, including acyl-coenzyme A synthase, apolipoproteins, and carnitine palmitoyltransferase 1 (Cpt1) (1, 3). PPAR{gamma} is most abundant in adipose tissue, and it is a critical transcription factor in the regulation of lipid storage and lipogenesis (1, 2, 3). Although PPARβ/{delta} is expressed in almost all tissues, the role of PPARβ/{delta} on metabolism has not been clarified (1). Synthetic PPAR{alpha} agonists (fibrate) and PPAR{gamma} agonists (thiazolidinediones) are in therapeutic use to treat dyslipidemia and diabetes (2, 11, 12). PPARs are also expressed in the brain, where they play an important role in neuroprotection (2, 13).

Previous studies have shown that changes in energy balance influence sleep/wake patterns (14). For example, high-fat feeding and food restriction paradigms that alter energy consumption can affect sleep/wake patterns. A high-fat diet leads to increases in the duration of nonrapid eye movement (NREM) sleep in mice (15), and alters the expression patterns of circadian clock genes (16). In addition, long-term food restriction increases sleep time during the active period (17). Interestingly, a recent study of humans showed the relationship between the risk of type 2 diabetes and slow-wave sleep, known as stages 3 and 4 in NREM sleep (18). Slow-wave sleep suppression, accompanied by reduced electroencephalogram (EEG) delta-oscillation (the dominant EEG frequency range in slow-wave sleep), decreased insulin sensitivity and glucose tolerance. Those findings suggest a close relationship between slow-wave sleep and energy metabolism.

We have recently found that PPAR{alpha} is involved with circadian clock control (19, 20, 21). The expression of PPAR{alpha} is tightly controlled by the Clock gene (20). Inversely, mice treated with bezafibrate, which is an antihyperlipidemic PPAR ligand, showed phase-advanced circadian locomotor activity (19). Homozygous Clock mutant mice, a useful model of delayed sleep phase syndrome (22, 23), also showed phase-advanced activity when given bezafibrate. Although fibrate might be useful for the treatment of delayed sleep phase syndrome (19), the effects of bezafibrate on sleep and body temperature (BT) remain to be elucidated. Therefore, we examined whether chronic treatment with bezafibrate would alter sleep and BT regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male ICR mice (Slc Inc., Shizuoka, Japan) were 8 wk of age at the beginning of the experiment. The mice were fed ad libitum and maintained on a 12-h light, 12-h dark cycle (lights on at 0900 h) at a controlled ambient temperature (24 ± 1 C). The experiments were approved by the Animal Study Committee of Tokushima University (No. 07079), and performed according to the guidelines for the Care and Use of Animals approved by the Council of the Physiological Society of Japan.

Feeding conditions
Mice (n = 7) were fed with a normal diet (MF; Oriental Yeast, Tokyo, Japan) for 2 wk, after which their feed was supplemented with 0.5% bezafibrate (Sigma Chemical Co., St. Louis, MO) for 2 wk. After that, the mice were returned to their normal diet for 5 wk.

Sleep recordings and analysis
Mice were anesthetized with a cocktail of ketamine (100 mg/kg) and xylazine 25 mg/kg). A telemetric device (TA10TA-F20; Data Sciences International, St. Paul, MN) for BT was implanted in the peritoneal cavity. Stainless steel miniature screw electrodes were implanted in the skull to record the EEG, and Teflon-coated stainless steel wires (Cooner wire, Chatsworth, CA) were implanted in the neck muscles on both sides to record the electromyogram (EMG). After 1 wk recovery, the mice were transferred to plastic cages (20 x 24 x 30 cm) in a soundproof recording room and allowed 2 d for habituation. The mice were connected by flexible cables to a polygraph and computer-assisted data acquisition system CED 1401 data processor (Cambridge Electronic Design, Cambridge, UK). Ten days after surgery, polygraphic recordings for BT, EEG, and EMG were collected continuously for 48 h under light-dark conditions, and data recorded for 24 h (the last 12 h of the first day and first 12 h of the second day) were analyzed.

Off-line sleep scoring was done on the computer screen by visual assessment of the EEG and EMG activities using the Spike2 analysis program (Cambridge Electronic Design). The vigilance states were classified by 6-sec epochs as wakefulness, rapid-eye movement (REM), or NREM sleep. NREM sleep was characterized by a continuous, slow, high-voltage EEG and low-voltage EMG activity. REM sleep was characterized by low-voltage EEG with continuous {theta}-waves and total suppression of the EMG. The EEG power spectrum in the epoch that was determined to be NREM sleep was calculated by Fast Fourier Transform using the Spike2 analysis program. The EEG {delta}-frequency band was set at 0.8–4.0 Hz. The delta-power was normalized as a percentage of the total power (0.8–50 Hz). BT and sleep/wake durations were averaged for hourly intervals. A single cosine wave was fitted by a least squares method for each mouse to evaluate the amplitude and acrophase (determined as a peak position of the cosine wave) of the BT and sleep/wake rhythms.

Spontaneous activity
Spontaneous physical activity was measured using an animal movement analysis system (ACTIMO System; Shintechno, Fukuoka, Japan), which consists of a rectangular enclosure (30 x 20 cm), with a sidewall equipped with photosensors at 2 cm intervals. Each pair of photosensors scanned animal movement at 0.5-sec intervals. Spontaneous activity of mice (n = 8) was recorded after treatment with normal diet for 2 wk, bezafibrate-supplemented diet for 2 wk, and normal diet for 5 wk. Mice were placed in the individual chambers and housed in these cages for 2–3 d before each recording to be familiarized with the recording environment. Movement signal counts were performed using the Spike2 analysis program.

Sleep deprivation (SD)
Mice in a separate group were fed a control diet (n = 5) or a bezafibrate-supplemented diet (n = 5) for 2 wk, during which time baseline recordings were performed. The following day, animals were sleep deprived for 6 h during the first half of the light phase by gentle handling. To keep an animal awake, an experimenter observed the EEG and EMG recordings for signs of sleep, and then touched the back of the mouse with a small soft brush several times. At the beginning of the last half of the light phase, SD was terminated, and the EEG and EMG were recorded for a 6-h period of uninterrupted recovery sleep.

DNA microarray analysis
Control and bezafibrate-treated mice were killed for molecular analysis at Zeitgeber time (ZT) 10, when BT showed no difference between the groups, and ZT22, when BT showed the largest difference between the groups. Each sampled group contained four mice. The square area containing the hypothalamus was dissected from the brain immediately after decapitation. The upper limit of the square area was about 4.5-mm below the surface of the skull, and the lateral limit of the square area was about ±1.5-mm to the midline. The anterior limit of the square area extended from the bregma, including the ventrolateral preoptic area, which is considered to be the NREM sleep center. The posterior limit of the square area was about 3 mm from the bregma, including the tuberomammillary nucleus, which is considered to be the histaminergic wake center. The tissue of the hypothalamus was stored in RNAlater stabilization reagent (QIAGEN, Hilden, Germany) for 2 h and then processed for molecular analyses (microarray and real-time RT-PCR). Total RNA was isolated following QIAGEN's RNA isolation protocol (RNeasy Mini kit; QIAGEN, Hilden, Germany). Contaminating DNA was removed using RNase-free DNase set (QIAGEN) during the process of RNA purification. The quality of the purified RNA applicable for microarray analysis was assessed by an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Labchip kit (Agilent Technologies, Inc., Palo Alto, CA). Equivalent amounts of total RNA from the hypothalamus of each of the four mice under the same conditions were mixed uniformly and then subjected to microarray analysis. Amplification and labeling of each RNA mixture were started with 400 ng total RNA using Low RNA Input Linear Amplification Kit PLUS and cyanine 3 CTP (Agilent Technologies). Hybridization of cyanine three-labeled cRNA with Whole Mouse Genome (4 x 44 K) Oligo Microarray (G4122F; Agilent Technologies) was performed following the manufacturer’s protocol. After washing, fluorescence intensity at each spot was assayed using a DNA Microarray scanner BA (G2565BA; Agilent Technologies). Signal intensities were quantified and analyzed by subtracting backgrounds using Feature Extraction Software Version 9.5 (Agilent Technologies). Data processing was performed using GeneSpring 7.3 software (Agilent Technologies). Data were transformed by setting all measurements less than 0.01 to 0.01. Data points that did not have detectable signals and those that represented microarray controls were labeled as absent, those representing either nonuniform or saturated features were labeled as marginal, and all remaining data points were labeled as present. All data points were median scaled using the median signal intensity value for data points labeled as present. Intensity data were linear-log transformed and assessed in scatter plots in which the intensity data from Bezafibrate-treated or nontreated mice were on the vertical axis or horizontal axis, respectively. The scatter plots were examined for the best number of percentile for the global normalization. All probe sets labeled as present and raw signals more than 30 in all arrays were retained after filtering. The remaining 22,999 probes were processed for the detection of genes whose expression levels had changed more than 2-fold or less than 0.5-fold in bezafibrate-treated mice compared with control mice at each ZT10 and ZT22 point.

Real-time RT-PCR
Total RNA from the hypothalamus used for DNA microarray analysis was reanalyzed by real-time RT-PCR. Total RNA in the liver was isolated with an acid guanidinium thiocyanate-phenol-chloroform mixture (ISOGEN; Nippon gene, Toyama, Japan). Real-time PCR quantification of target mRNAs was performed as described previously (24). cDNA was generated from 0.5 µg (hypothalamus) or 1.0 µg (liver) of each total RNA sample using a GeneAmp RNA PCR kit (Applied Biosystems, Foster City, CA). We used predesigned, gene-specific TaqMan probes and primer sets to assess expression of the following genes: adenosine A2A receptor (Adora2a; Mm00802075_m1), neuropeptide Y (NPY) (Npy; Mm00445771_m1), proopiomelanocortin (POMC)-{alpha} (Pomc1; Mm00435874_m1), fibroblast growth factor 21 (Fgf21; Mm00840165_g1), pancreatic colipase (Clps; Mm00517960_m1), pancreatic lipase-related protein 2 (Pnliprp2; Mm00448214_m1), Cpt1a (Mm00550438_m1), and 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (Hmgcs2; Mm00550050_m1). The real-time PCR was performed with an Applied Biosystems 7900HT real-time PCR system using TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instruction. β-Actin (Actb; Mm00607939_s1) was used for endogenous quantity control, and values were normalized to β-actin mRNA expression.

Measurement of plasma ketone level
Trunk blood was collected from each group at decapitation for DNA microarray and real-time RT-PCR analysis. Plasma ketone bodies, acetoacetate, and 3-hydroxy-butyrate were measured enzymatically using an automatic analyzer system JCA-BM12 (JEOL, Tokyo, Japan) and reagents for measurement of ketone bodies by enzymatic assay (Kainos Laboratories, Tokyo, Japan).

Pharmacological treatments and injection procedure
A 25-gauge guide cannula was stereotaxically implanted into the lateral ventricle according to the following coordinates: 0.46-mm posterior to the bregma, 1.20-mm lateral to the midline, and 2.00-mm below the surface of the skull. The cannula was fixed to the skull with screw electrodes for EEG recordings. A 31-gauge dummy cannula, cut within the end of the guide cannula, was inserted into the guide shaft to maintain cannula patency. After surgery, mice were caged individually and allowed to recover for 10 d, during which they were habituated to experimental handling. Mice treated with bezafibrate for 2 wk were injected with 1229U91 (GR231118; Sigma Chemical), an NPY Y1 receptor antagonist (NPY Y1A), or vehicle at ZT11.5. NPY Y1A (1.7 nmol dissolved in 1.0 µl artificial cerebrospinal fluid) or vehicle was injected slowly over 1 min using a Hamilton microsyringe connected with a 31-gauge injection cannula. The injection cannula was then left in place for 5 min to minimize reflux before it was removed.

To check the ceiling effect of delta-power in NREM sleep, we used histamine H1 receptor antagonist (H1A), pyrilamine (Sigma Chemical), which is known to induce NREM sleep. Pyrilamine (5 and 50 mg/kg) and vehicle (saline) were injected ip into normal mice at ZT20.5. EEG recording started at ZT21 and delta-power in NREM sleep for the first 1 h after the drug injections was analyzed.

Statistics
Results are expressed as the means ± SEM. The changes of BT, sleep architecture, and EEG delta-power were analyzed by repeated measures of one-way or two-way ANOVA, followed by a Tukey’s post hoc test. The data of real-time RT-PCR analyses and plasma ketone levels were analyzed by the Mann-Whitney U test. In a few instances, such as the effect of SD and NPY Y1A injection, paired t tests were used for comparisons between the same mice. P < 0.05 was taken as an indication of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BT
Two weeks of treatment with bezafibrate decreased body weight compared with aged-matched controls fed a normal diet (normal, 42.67 ± 0.74 g; bezafibrate, 35.41 ± 0.55 g; P < 0.001). We compared the rhythm of BT among control and treated mice during and after bezafibrate treatment. There were significant differences in the shape of the rhythms among these feeding conditions [groups x time; F(2, 46) = 3.03; P < 0.001; Fig. 1AGo]. When bezafibrate was administered, BT showed a larger decrease in the latter half of the dark period than that of the control and after recovery. The averaged 6-h BT in the latter half of the dark period [Dark 2, F(2, 16) = 11.51; P < 0.001] and the first half of the light period [Light 1, F(2, 16) = 4.50; P < 0.05] was lower in bezafibrate-treated conditions than controls (Fig. 1BGo). There was no significant change in the amplitude of BT rhythm by bezafibrate treatment (Fig. 1CGo). The acrophase of rhythm for BT was advanced about 2 h after bezafibrate treatment in comparison with the controls [F(2, 16) = 11.06; P < 0.001; Fig. 1DGo]. The advanced acrophase of BT caused by bezafibrate returned to baseline values after 5 wk feeding the control diet.


Figure 1
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  		FIG. 1. The effect of bezafibrate on BT. A, Time course of BT in mice fed a control diet (Con) (open circles), bezafibrate-supplemented (Fib) diet (filled circles), or the control diet after recovery (Rec) (gray circles). B, BT changes in mice fed the control diet (open columns), bezafibrate-supplemented diet (filled columns), or control diet after recovery (hatched columns) in ZT0–5 (Light 1), ZT6–11 (Light 2), ZT12–17 (Dark 1), and ZT18–23 (Dark 2). Amplitude (C) and acrophase (D) of BT rhythms. *, P < 0.05; **, P < 0.01, bezafibrate vs. control; {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01, bezafibrate vs. recovery (Tukey’s post hoc test). Data represent means ± SEM (n = 5–7).

 
Sleep/wake rhythm and EEG delta-oscillation
Sleep architecture and sleep/wake rhythm were also affected by 2 wk bezafibrate treatment (Fig. 2Go, A–C). Over 24 h, the duration of wakefulness, NREM, and REM sleep were not changed by bezafibrate feeding (Fig. 2Go, D–F). However, in the light period, bezafibrate treatment significantly increased the duration of wakefulness [F(2, 17) = 5.86; P < 0.05] and decreased the duration of NREM sleep [F(2, 17) = 8.58; P < 0.01] compared with the recovery period (Fig. 2Go, D and E). Bezafibrate did not affect the amplitude of the sleep/wake rhythm (Fig. 3Go, A–C). The acrophase of rhythm for wakefulness [F(2, 17) = 13.61; P < 0.001] and NREM sleep [F(2, 17) = 16.57; P < 0.001] were advanced about 3 h after bezafibrate treatment in comparison with the control (Fig. 3Go, D and E). The bezafibrate-induced advancement of the acrophase of the sleep/wake rhythm returned to control values after the recovery period. The hourly averaged power of the EEG delta-oscillation in NREM sleep differed significantly between control and bezafibrate [groups x time; F(2, 46) = 1.69; P < 0.01; Fig. 4AGo]. In the bezafibrate-treated mice, the averaged EEG delta-power for 24 h (normalized by control period) was significantly higher than the control and recovery period [F(2, 17) = 8.12; P < 0.01; Fig. 4BGo]. The amplitude of delta-power rhythm in NREM sleep was decreased by bezafibrate compared with the recovery period [F(2, 17) = 5.03; P < 0.05], whereas the acrophase of delta-power rhythm was not changed by bezafibrate (Fig. 4Go, C and D). The hourly averaged spontaneous locomotor activity was not changed by bezafibrate treatment (data not shown).


Figure 2
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  		FIG. 2. Sleep/wake duration in each feeding condition. Time course for changes in wakefulness (A), NREM sleep (B), and REM sleep (C) in mice fed a control diet (open circles), bezafibrate-supplemented diet (filled circles), and control diet after recovery (gray circles). Total duration of wakefulness (D), NREM sleep (E), and REM sleep (F) in mice fed a control diet (open columns), bezafibrate-supplemented diet (filled columns), or control diet after recovery (hatched columns). **, P < 0.01, recovery vs. control; {dagger}{dagger}, P < 0.01, bezafibrate vs. recovery (Tukey’s post hoc test). Data represent means ± SEM (n = 6–7).

 

Figure 3
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  		FIG. 3. Amplitudes and acrophases of sleep/wake rhythms. Amplitudes of rhythms for wakefulness (A), NREM sleep (B), and REM sleep (C) in mice fed a control diet (Con) (open columns), bezafibrate-supplemented diet (Fib) (filled columns) or control diet after recovery (Rec) (hatched columns). Acrophase of rhythms for wakefulness (D), NREM sleep (E), and REM sleep (F) in mice fed a control diet (open circles), bezafibrate-supplemented diet (filled circles), and control diet after recovery (gray circles). *, P < 0.05, bezafibrate vs. control; {dagger}{dagger}, P < 0.01, bezafibrate vs. recovery (Tukey’s post hoc test). Data represent means ± SEM (n = 6–7).

 

Figure 4
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  		FIG. 4. Bezafibrate treatment increased EEG delta-power in NREM sleep. A, Time course of EEG delta-power in NREM sleep in mice fed a control diet (Con) (open circles), bezafibrate-supplemented diet (Fib) (filled circles), and control diet after recovery (Rec) (gray circles). B, Averaged EEG delta-power for 24 h in mice fed a control diet (open column), bezafibrate-supplemented diet (filled column), or control diet after recovery (hatched column). Amplitudes (C) and acrophases (D) of rhythms for EEG delta-power. **, P < 0.01, bezafibrate vs. control; {dagger}, P < 0.05, {dagger}{dagger}, P < 0.01, bezafibrate vs. recovery (Tukey’s post hoc test). Data represent means ± SEM (n = 6–7).

 
Rebound response after SD
The homeostatic responses after 6 h SD were assessed in a separate group fed the indicated diets. Mice treated with bezafibrate tended to increase the length of wakefulness and decrease NREM sleep duration compared with the control diet during the recovery period after SD (Fig. 5AGo). In mice treated with the control diet, NREM delta-power was significantly increased in the first 3 h of the recovery period compared with baseline [groups x time; F(1, 5) = 15.47; P < 0.001; Fig. 5BGo]. In contrast, bezafibrate-treated mice showed a higher baseline level and no significant rebound in delta-power in NREM sleep after SD (Fig. 5CGo).


Figure 5
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  		FIG. 5. The rebound response of EEG delta-power after SD was attenuated by bezafibrate treatment. A, The change of sleep/wake duration for 6 h after SD. Data are expressed as the percent change from the baseline value at the same time of the preceding day in the mice fed the control diet (open column) and bezafibrate-containing diet (filled column). Time course of EEG delta-power for baseline (open circles) and after SD (filled circles) in mice fed control diet (B) and bezafibrate-supplemented diet (C). *, P < 0.05; **, P < 0.01, vs. baseline (paired t tests). Data represent means ± SEM (n = 5).

 
Fgf21, pancreatic lipases, ketogenic enzymes in the liver and ketone levels in the plasma
Fgf21 is induced directly by PPAR{alpha} in the liver in response to fasting and PPAR{alpha} agonists (7, 8, 25). Fgf21 in turn stimulates lipolysis and ketogenesis. Clps and Pnliprp2, pancreatic lipases, are increased dramatically by PPAR{alpha} agonists, although their genes are normally expressed at very low levels in the liver (8). Cpt1a and Hmgcs2 are also regulated by PPAR{alpha}, and they mediate crucial steps in ketogenesis. To check the efficacy of bezafibrate on the activation of PPAR{alpha}, we measured mRNA levels of Fgf21, Clps, Pnliprp2, Cpt1a, and Hmgcs2 in the liver. Plasma ketone levels were also assessed. Bezafibrate treatment significantly increased the expression of Fgf21, Clps, and Hmgcs2 mRNAs at both ZT10 and ZT22, and Cpt1a and Pnliprp2 mRNA at ZT22 in the liver (Fig. 6Go, A–E). Plasma acetoacetate acid levels were increased at both ZT10 and ZT22 by bezafibrate, whereas β-hydroxybutyrate levels were decreased (Fig. 6FGo). There was no significant difference in total ketone levels between the two groups, although the balance between acetoacetate and β-hydroxybutyrate was significantly changed.


Figure 6
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  		FIG. 6. The effect of bezafibrate on PPAR{alpha}-inducible genes relating with lipolysis and ketogenesis. The expression of Fgf21 (A), Clps (B), Pnliprp2 (C), Cpt1a (D), and Hmgcs2 (E) mRNAs in the liver and plasma concentrations of ketone bodies (F) were measured in mice fed a control diet (open columns) or the bezafibrate-supplemented diet (filled columns). *, P < 0.05; **, P < 0.01, bezafibrate vs. control (Mann-Whitney U test). Data represent means ± SEM (n = 4–5 per column).

 
DNA microarray analysis and real-time RT-PCR in the hypothalamus
To determine the effect of bezafibrate treatment on gene expression in the brain, we measured mRNA levels in the hypothalamus, the region primarily responsible for regulating BT and sleep. DNA microarray revealed 19 up-regulated genes and 58 down-regulated genes at ZT10, and 37 up-regulated genes and 66 down-regulated genes at ZT22 that had at least a 2- or 0.5-fold change. We extracted genes that showed raw signal intensities greater than 1000 in control or bezafibrate-treated groups (Table 1Go). Only a few genes, Adora2a, Npy, and Pomc1 are known to be involved in sleep regulation or energy metabolism. Real-time RT-PCR analysis confirmed the microarray data, and demonstrated a significant increase of Npy at both ZT10 and ZT22 (Fig. 7BGo), and a decrease of Pomc1 at ZT10 (Fig. 7CGo).


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  		TABLE 1. The list of bezafibrate-induced up-regulated and down-regulated transcripts in the hypothalamus by DNA microarray analysis

 

Figure 7
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  		FIG. 7. Real-time RT-PCR analysis of gene expression in the hypothalamus at ZT10 and ZT22. Expression of Adora2a (A), Npy (B), and Pomc1 (C) mRNAs was measured in mice fed a control diet (open circles) or a bezafibrate-supplemented diet (filled circles). *, P < 0.05; **, P < 0.01, bezafibrate vs. control (Mann-Whitney U test). Data represent means ± SEM (n = 4 per group).

 
Intracerebroventricular (icv) injection of NPY Y1A
NPY is known to be involved with torpor-like hypothermia in rodents (26, 27). We investigated the effects of NPY on the decrease of BT and the increase of delta-power caused by bezafibrate. Thus, mice were treated with bezafibrate for 2 wk and received central administration of NPY Y1A, which reportedly prevents NPY-induced torpor-like hypothermia (27). After the injection, there was a significant difference in BT between NPY Y1A- and vehicle-treated mice [groups x time; F(1, 11) = 5.82; P < 0.001; Fig. 8Go]. The BT decrease observed in bezafibrate-treated mice in the latter half of the dark period was not observed when NPY Y1A was administrated. On the other hand, there was no difference between the groups in EEG delta-oscillations during NREM sleep for 2 h before and after the drug injection, and 2 h at the end of the dark phase.


Figure 8
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  		FIG. 8. The effect of icv administration of NPY Y1A (filled circles) or vehicle (open circles) on BT in mice fed the bezafibrate-supplemented diet. *, P < 0.05, vs. vehicle (paired t tests). Data represent means ± SEM (n = 5). EEG delta-power in NREM sleep in mice treated with NPY Y1A (filled columns) or vehicle (open columns) was indicated with an average of 2 h at ZT10–11 (left), ZT12–13 (middle), and ZT23–24 (right). Data represent means ± SEM (n = 6). Arrow indicates injection time.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings herein show that chronic bezafibrate treatment decreased BT during the latter half of the dark period with phase advancement and increased EEG delta-power in NREM sleep, which showed no rebound after 6 h SD.

The hypothalamus, particularly the arcuate nucleus, plays a key role in the integration of signals for nutrient availability and energy balance, including metabolic and feeding control. The two major pathways that include NPY and POMC regulate food intake and reside in the arcuate nucleus (28). In mice the NPY pathway is critical for both the full torpor phenotype and the modulation of torpor by ghrelin, whereas the POMC pathway is not required for the torpor (26). In the present study, the hypothalamus of bezafibrate-treated mice showed significantly increased expression of Npy mRNA and decreased Pomc1 mRNA. Furthermore, icv injection of Y1A diminished bezafibrate-mediated decreases in BT. Therefore, it is possible that bezafibrate treatment decreased BT during the latter half of the dark period via activation of NPY-Y1 receptor signaling.

The mechanism underlying decreased BT could be related to the PPAR{alpha}-Fgf21 pathway. PPAR{alpha} promotes hepatic fatty acid oxidation and ketogenesis in response to fasting, and Fgf21 is induced directly by PPAR{alpha} in the liver in response to PPAR{alpha} agonist and fasting (7, 8, 9, 25). In fact, in our study, significant increases of hepatic Fgf21, Clps, Pnliprp2, Cpt1a, and Hmgcs2 mRNA levels were observed, indicating the significant effect of bezafibrate on the PPAR{alpha}-Fgf21 pathway. Decreased core BT in the latter half of the dark period in response to fasting was observed in mice that had transgenic overexpression of Fgf21 or were treated with PPAR{alpha} agonist (8). In the present study, the bezafibrate-induced BT decrease was evident in the latter half of the dark period and was quite similar to Fgf21-mediated decreases in BT (8). These data may suggest that bezafibrate, a PPAR pan agonist, could activate the PPAR{alpha}-Fgf21 pathway and, thereby, decrease BT. Because hypothalamic NPY has already been reported to be increased by fasting (29), a fasting-like state linked with activation of the PPAR{alpha}-Fgf21 pathway may cause up-regulation of NPY and then a decrease in BT.

Central administration of NPY affects not only BT but also circadian rhythm. Microinjection of NPY into the suprachiasmatic nuclei (SCNs) during the middle of the subjective day induces phase advancement of the circadian clock, whereas having little effect during subjective night (30, 31, 32). NPY attenuated light-induced phase delays and inhibited phase advances, suggesting that NPY in the SCNs plays an active role in both photic and nonphotic effects on the circadian clock (32, 33). In the present study, bezafibrate increased Npy expression in the hypothalamus, which might affect circadian rhythm. However, the expression of genes critical to the circadian system such as Per1 (NM_011065; ZT10: 1.03, ZT22: 1.15) and Per2 (NM_011066; ZT10: 1.04, ZT22: 1.09) did not change in the DNA microarray. These results coincide with our previous report that bezafibrate did not affect the circadian expression of Per2 mRNA in the SCNs but advanced Per2 expression in the cortex and liver as determined by in situ hybridization and Northern blotting (19). Thus, it is possible that bezafibrate affects not the SCNs but peripheral oscillators. In the present study, bezafibrate treatment decreased BT during the latter half of the dark period, and this change of diurnal profile could have resulted in the advancement of the acrophase of BT rhythm. We will explore icv injections of PPARs agonist to determine whether decreased BT and phase advancement are central or peripheral effects.

Sleep/wake rhythm and depth of sleep are thought to be determined by a circadian and homeostatic process, which is modeled as a "two process model of sleep regulation" (34). The timing of sleeping and awakening is critically under the control of the circadian system. On the other hand, the homeostatic process of sleep is thought to be regulated independently of circadian timing. It is believed that sleep debt (or sleep pressure) increases during wakefulness and dissipates during sleep. The power of the EEG delta-oscillation has been used as physiological markers of sleep depth and homeostatic need for sleep (35). In fact, EEG delta-oscillation in NREM sleep increased during the dark period, which is the active period for mice, and decreased during the light period, which is their rest period in our study (Fig. 4AGo). In both human and experimental animals, it is well known that prolonged wakefulness or SD causes a large increase in the EEG delta-power. This homeostatic mechanism of sleep is thought to have an essential role for ensuring rest and maintenance of neural function. It is hypothesized that homeostatic regulation of the EEG delta-power in sleep is linked to synaptic potentiation and downscaling (36, 37). The present study has shown that a PPAR agonist increases the EEG-delta oscillation in NREM sleep without affecting the duration of wakefulness or spontaneous activity. It is possible that PPARs are directly involved in the delta-oscillation mechanism.

PPARs form heterodimers with RXRs and bind to specific DNA sequences, the PPARs responsive element, thereby regulating the expression of target genes involved in lipid and glucose metabolism (1, 2, 4, 10). The RXRs bind to 9-cis-retinoic acid, which is a derivative of vitamin A. Recently, we reported that deficiency of vitamin A induces a decrease in EEG delta-power in NREM sleep (38). However, in the case of vitamin A deficiency, spontaneous activity was suppressed, and SD recovered the EEG delta-power. The decreased EEG delta-power in mice with vitamin A deficiency may be secondary and caused by changes of activity or state in wakefulness. This suggests that retinoid-related functions may not be directly involved with delta-oscillation. The mice treated with bezafibrate showed no rebound of NREM delta-power after 6 h SD, whereas mice treated with the control diet did show a large increase of NREM delta-power. It is reported that not only delta-power but also duration of NREM sleep are increased by SD in normal mice (39, 40). After SD the increased duration of NREM sleep seen in mice on a normal diet was attenuated in the bezafibrate-treated mice, although the difference did not reach a significant level. It is noteworthy that bezafibrate-treated mice have an increased baseline level of EEG delta-power that is close to the level after SD in the control mice. The chronically increased delta-power caused by bezafibrate would induce a tolerance for SD and cause the absence of the rebound of NREM delta-power after SD. Because an icv injection of Y1A did not modify the EEG delta-power in bezafibrate-treated mice, the NPY-Y1 receptor is probably not involved in the increase of EEG delta-power. It is then indicated that the mechanism in increased EEG delta-power by PPAR activation is independent of that in decreased BT.

It is possible that the absence of a rebound response of EEG delta-oscillation in the bezafibrate-treated mice is a ceiling effect resulting from the high level of EEG delta-power at baseline. To check whether the EEG delta-power could be increased to a higher level than that seen in bezafibrate-treated mice or after SD in control diet-treated mice (~30% in Fig. 5Go, B and C), H1A [a well-known promoter of NREM sleep (41)] was injected into normal mice. The injection of high concentrations of H1A significantly increased delta-power in NREM sleep compared with vehicle and low concentrations [F(2, 9) = 17.14; P < 0.001; Fig. 9Go]. Mice injected with high concentrations of H1A showed a high level of delta-power at 37.2–48.9% (average: 41.5), suggesting that EEG delta-power could be even higher than that of bezafibrate-treated mice. EEG delta-power in mice injected with low concentrations of H1A showed 27.8–31.6% (average: 29.5), similar to the baseline level in bezafibrate-treated mice or after SD in control diet-treated mice. These data suggest that an absence of rebound response in EEG delta-power observed in the bezafibrate-treated mice was not due to a physiological and mechanical ceiling effect, but it could be a meaningful effect for roles of PPARs in sleep homeostatic regulation.


Figure 9
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  		FIG. 9. High concentration (50 mg/kg; filled column) of H1A (ip) increased EEG delta-power in NREM sleep compared with vehicle (Veh) (open column) and low concentration (5 mg/kg; hatched column). **, P < 0.01, vs. vehicle; {dagger}{dagger}, P < 0.01, vs. H1A 5 mg/kg (Tukey’s post hoc test). Data represent means ± SEM (n = 4).

 
Activation of PPAR{alpha} with fasting induces ketogenesis and increases plasma total ketone bodies. Higher levels of ketone bodies affect neural activity (42). However, in our study the total ketone body level was not increased. Therefore, changes of ketogenesis caused by PPAR{alpha} activation are probably not the cause of the increase in EEG delta-power. At present, the neural mechanism of enhancement of EEG delta-oscillation remains unclear. Additional experiments are necessary to clarify the role of PPARs in the homeostatic control of sleep.

In conclusion, chronic bezafibrate treatment decreased BT and enhanced delta-oscillation during sleep with a loss of rebound after 6 h SD. Nuclear receptor PPARs could play a significant role in the interaction between lipid/glucose metabolism and sleep homeostasis and/or BT regulation.


   Acknowledgments
 
We thank Shigenobu Shibata (Waseda University, Tokyo, Japan) for helpful discussions.


   Footnotes
 
This work was supported by a Grant-in-Aid (18603005) (to H.S.) from the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research from the 21st Century Center of Excellence Program, Human Nutritional Science on Stress Control, Tokushima, Japan.

Database accession nos.: GSE10417, GSM262509, and GSM262510.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 11, 2008

Abbreviations: BT, Body temperature; Cpt1, carnitine palmitoyltransferase 1; EEG, electroencephalogram; EMG, electromyogram; H1A, histamine H1 receptor antagonist; icv, intracerebroventricular; NPY, neuropeptide Y; NPY Y1A, neuropeptide Y Y1 receptor antagonist; NREM, nonrapid eye movement; POMC, proopiomelanocortin; PPAR, peroxisome proliferator-activated receptor; REM, rapid-eye movement; RXR, retinoid X receptor; SCN, suprachiasmatic nucleus; SD, sleep deprivation; ZT, Zeitgeber time.

Received February 29, 2008.

Accepted for publication June 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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