Cholesterol diet enhances daily rhythm of Pai-1 mRNA in the mouse liver
Takashi Kudo,
Emiko Nakayama,
Sawako Suzuki,
Masashi Akiyama, and
Shigenobu Shibata
Department of Pharmacology, School of Science and Engineering, Waseda University, Tokyo 202-0021, Japan
Submitted 26 February 2004
; accepted in final form 2 June 2004
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ABSTRACT
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Myocardial infarction frequently occurs in the morning, a phenomenon in part resulting from the downregulation of fibrinolytic activity. Plasminogen activator inhibitor-1 (PAI-1) is a key factor behind fibrinolytic activity, and its gene expression is controlled under the circadian clock gene in the mouse heart and liver. Hypercholesterolemia has been associated with impaired fibrinolysis due to enhanced PAI-1 activity, which has also been implicated in atherosclerosis. The aim of this study was to decipher whether the Pai-1 gene is still expressed daily with hypercholesterolemia. Hypercholesterolemia (1% cholesterol diet) did not significantly affect the daily expression of clock genes (Per2 and Bmal1) and clock-controlled genes (Dbp and E4bp4) in the liver (P > 0.05); however, daily expression of the Pai-1 gene and Pai-1 promoter regulating factor genes such as Nr4a1 was significantly upregulated (P < 0.01). Daily restricted feeding for 4 h during the day reset the gene expression of Per2, Pai-1, Nr4a1, and Tnf-
. Lesion of the suprachiasmatic nucleus, the location of the main clock system, led to loss of Per2 and Pai-1 daily expression profiles. In the present experiments, we demonstrated that hypercholesterolemia enhanced daily expression of the Pai-1, Tnf-
, and Nr4a1 genes in the mouse liver without affecting clock and clock-controlled genes. Therefore, the risk or high frequency of acute atherothrombotic events in the morning still seems to be a factor that may be augmented under conditions of hypercholesterolemia.
circadian; clock gene; hypercholesterolemia; suprachiasmatic; fibrinolytic
REPORTS INDICATE THAT the onset of heart failure and sudden death is greatest in the morning (17, 18). This phenomenon is partly a result of downregulated fibrinolytic activity. Plasminogen activator inhibitor-1 (PAI-1) activity, a key factor in fibrinolytic activity, shows a circadian change in humans that is high in the morning and low in the evening (1, 10). Thus the circadian changing of PAI-1 activity may help to explain why myocardial infarction frequently occurs from 6 AM to 12 PM (18). Because Pai-1 mRNA can be found in adipocytes, the liver, the kidneys, and abundantly in the heart (21), PAI-1 supplied by these organs may facilitate fibrinolysis. Recent investigations have demonstrated a circadian change in Pai-1 mRNA both in vivo and in vitro (11, 14) as well as the upregulation of Pai-1 by BMAL2-CLOCK and BMAL1-CLOCK through E-box sites (9) and downregulation by PER2 and CRY1 in vitro (11, 22). Although peripheral clock systems may be involved in the circadian expression of Pai-1 mRNA in the peripheral organs, we still do not know whether environmental stimuli-induced Pai-1 gene expression is controlled by the clock system.
Alterations in plasma lipid levels exert an important impact on vascular function, because they can affect several endothelial cell processes. In this regard, numerous studies (3, 5, 31) have shown a link between hypercholesterolemia and functional endothelial alterations. Hypercholesterolemia in humans has been associated with impaired fibrinolysis resulting from either reduced tissue-type plasminogen activator or enhanced PAI-1, both of which also participate in the complication of atherosclerosis (7, 20). On the other hand, in a study (13) on fatty diets and Pai-1 expression, a high-fat diet had no effect on Pai-1 circadian rhythm, although this was in humans and over a much shorter period.
The present investigation was therefore conducted in part to elucidate whether hypercholesterolemia causes a robust daily expression or sustained high expression of the Pai-1 gene in the mouse liver. In addition, we examined the effect of hypercholesterolemia on the expression of clock genes such as Per2 and Bmal1 and clock-controlled genes such as Dbp (28, 29) and E4bp4 (15) in the mouse liver to determine whether Pai-1 gene expression is still controlled under clock function even when Pai-1 is upregulated by hypercholesterolemia.
PAI-1 synthesis is regulated by several second messenger signaling pathways such as TNF-
and NR4A1 (6, 8, 24). Therefore, we also examined whether hypercholesterolemia- induced high expression of the Pai-1 gene was associated with Nr4a1, Tnf-
, and/or Vldlr expression.
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MATERIALS AND METHODS
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Animals and diet.
Male ICR mice (6 wk old) were purchased through Takasugi Animal (Saitama, Japan). Animals were maintained on a light-dark cycle (12-h light, 12-h dark with lights on at 8:30 AM, room temperature of 23°C) and given food and water ad libitum except during restricted feeding (RF) experiments. To examine the aging effect on gene expression, we cared for mice until they reached 3, 6, or 12 mo of age under normal conditions. In this experiment, we used zeitgeber time (ZT; ZT0 was the lights-on time and ZT12 was the lights-off time) to define the clock time. For 6 wk, control mice were fed a standard mouse diet, and cholesterol-fed mice received a diet supplemented with 1% (wt/wt) cholesterol and 0.5% (wt/wt) cholic acid. All animal experiments were carried out in accordance with the Law (No. 105) and Notification (No. 6) of the Japanese Government and with Waseda University Human Science School Guidelines for the Care and Use of Laboratory Animals.
RNA isolation and RT-PCR.
Mice were deeply anesthetized with ether, and the livers and hearts were rapidly isolated, frozen in liquid nitrogen, and stored at 80°C until RNA isolation. Total RNA was extracted using ISOGEN Reagent (Nippon Gene; Tokyo, Japan). The DNA digestion step was performed using RQ1 RNase-Free DNase (Promega) after an incubation step at 37°C for 30 min. The enzyme was inactivated by RQ1 DNase Stop Solution (Promega). We used a semiquantitative RT-PCR method for measuring the expression level of mRNA. One hundred nanograms of total RNA were reverse transcribed and amplified using the Superscript One-Step RT-PCR System (Invitrogen) in a GeneAmp PCR System 9700 (Applied Biosystems). Specific primer pairs were designed based on the following published data on Per2, Bmal1, Dbp, E4bp4, Tnf-
, Vldlr, Pai-1, and
-actin genes in GenBank. Table 1 outlines the primer pairs used to amplify each primer product. PCR was executed under the following conditions: cDNA synthesis at 50°C for 30 min followed by 94°C for 2 min and PCR amplification for 2630 cycles with denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 68°C for 1 min. All PCR products were under the process of linear amplification from 26 to 30 cycles depending on the primer sets (
-actin, Per2, Bmal1, Dbp, E4bp4, Tnf-
, Vldlr, and Pai-1 cDNA were amplified for 28, 28, 28, 26, 29, 30, 28, and 28 cycles, respectively). The
-actin primer spanned exon borders 45 and 56, respectively, the Per2 primer spanned exon borders 34, 45, and 56, respectively, the Bmal1 primer spanned exon borders 12, 23, 34, 45, and 56, respectively, the Dbp primer spanned exon borders 23, the Tnf-
primer spanned exon borders 12, 23, and 34, respectively, the Vldlr primer spanned exon borders 1718 and 1819, respectively, and the Pai-1 primer spanned exon borders 34, 45, 56, 67, and 78, respectively, to ensure cDNA specificity of amplification reaction. From the 32nd cycle, product levels plateaued (data not shown). PCR products were electrophoresed on a 3% agarose gel stained with ethidium bromide and analyzed using an EDAS-290 system (Kodak). The intensity of the PCR product of the target gene was normalized to the intensity of
-actin. Reproducibility of the amplitude (ratio of peak to trough) and phase, as determined by this method (25), suggested that under the present experimental conditions we could adequately detect a circadian change in clock gene expression in the mouse liver.
RF experimentation.
RF was conducted as previously described (26). In brief, after 1 day of fasting (termed day 0), the daytime RF group of mice was allowed access to food for 4 h from ZT5 to ZT9 for 14 consecutive days. Water was continuously available throughout all experiments. The nighttime RF group was allowed to feed for 4 h from ZT17 to ZT21. Animals were killed at ZT1, -7, -13, and -19 on day 14.
Assay for serum and hepatic cholesterol levels.
Blood taken from each mouse (250750 µl) was mixed with 1.5 mg of potassium EDTA, and the plasma was obtained by centrifugation. A plasma sample (20 µl) from each mouse was used to obtain total cholesterol content with a cholesterol E-test kit (Wako Pure Chemical Industries; Osaka, Japan). The cholesterol content in the liver was measured as follows: 0.2 g of liver tissue from each mouse was homogenized in 4 ml of chloroform-methanol [2:1 (vol/vol)], after which 0.8 ml of 50 mM NaCl was added. A sample (50 µl) of the organic phase was mixed with 7.5 mg of Triton X-100. After evaporation of the organic solvents, the lipid in the detergent phase was used to measure total cholesterol content with a cholesterol E-test kit.
Suprachiasmatic nucleus lesion.
Bilateral thermal lesion of the suprachiasmatic nucleus (SCN) was performed as described in Wakamatsu et al. (26). A lesion was made by maintaining a temperature of 55°C for 15 s via a current path. After recovery from anesthesia, animals were moved to a locomotor activity device. Two weeks after surgery, we selected animals with complete lesioning of the SCN once arrythmicity was confirmed using a
2 periodogram (23) in the range of 2028 h. Twelve of the twenty SCN-lesioned animals with a confirmed loss of rhythmic activity were used for this study. Lesion sites were confirmed histologically at the end of the experiment.
Statistical analysis.
The values are expressed as means ± SE. For statistical analysis, one-way or two-way ANOVA was applied followed by the Fisher protected least-significant difference test or Student's t-test.
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RESULTS
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Daily expression of Pai-1 gene in the liver and heart under hypercholesterolemic conditions.
A cholesterol diet markedly increased the serum levels of total cholesterol from 104 ± 13 to 166 ± 12 mg/dl (P < 0.01, Student's t-test) and total hepatic cholesterol from 2.5 ± 0.2 to 15.9 ± 3.4 mg/g tissue (P < 0.01, Student's t-test). A diet supplemented with 0.5% (wt/wt) folic acid did not elevate total cholesterol in the serum or Pai-1 gene expression in the liver (data not shown).
With a normal diet, daily patterns of the Pai-1 gene clearly peaked in the evening in the heart (F3,20 = 9.1, P < 0.01, one-way ANOVA) but not in the liver (F3,20 = 1.1, P > 0.05, one-way ANOVA) (Fig. 1). On the other hand, after 6 wk on a cholesterol diet, the Pai-1 gene exhibited a clear daily expression pattern in the liver with a peak at ZT13 (F3,20 = 17.8, P < 0.01, one-way ANOVA) (Fig. 1, A and B). When a normal diet was compared with a cholesterol diet, two-way ANOVA revealed significant differences in Pai-1 expression (ZT x diet interaction) in the liver (F3,40 = 16.6, P < 0.01) but not in the heart (F3,40 = 0.46, P > 0.05). Total cholesterol content in the liver did not exhibit a daily pattern under either a normal or cholesterol diet, and total cholesterol content was significantly higher in the cholesterol diet group than in the normal diet group (F1,40 = 117, P < 0.01, diet effect, one-way ANOVA) (Fig. 1C).

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Fig. 1. Daily fluctuation of Pai-1 mRNA in the liver and heart of mice fed a normal diet or cholesterol diet ad libitum. A: representative electrophoresis photographs of PCR products from the two [normal diet (ND) and cholesterol diet (CD)] groups at zeitgeber time (ZT)1, -7, -13, and -19. The top open and solid bars represent light and dark periods, respectively. B: daily expression of the Pai-1 gene was plotted relative to the mRNA level, which was normalized to -actin expression (n = 6). The lowest level of relative mRNA was set as 100; therefore, each column exhibits the percent change from this set level. **P < 0.01 vs. normal diet group (two-way ANOVA followed by Student's t-test); ##P < 0.01 and #P < 0.05 vs. the ZT1 group [one-way ANOVA followed by Fisher protected least significant difference (PLSD) test]. C: daily content of total cholesterol in the liver was plotted. **P < 0.01 vs. normal diet group (two-way ANOVA followed by Student's t-test).
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Aging effect on hypercholesterolemia-induced Pai-1 gene expression in the liver.
Three-, six-, and twelve-mo-old mice were fed a cholesterol diet for 2 wk and then killed at ZT13, the time at which there was an obvious difference in Pai-1 gene expression between the normal and cholesterol diet groups (Fig. 1). Two-way ANOVA revealed significant differences in Pai-1 expression (aging x diet interaction) in the liver of cholesterol-fed mice and their groups (F2,12 = 4.5, P < 0.05) (Fig. 2B).Pai-1 gene expression in the liver was dependent on the age of the animal; the highest expression was found in 12-mo-old mice (P < 0.01 vs. 3 mo old, Student's t-test). Although cholesterol content in the liver significantly increased with cholesterol intake for all three ages (P < 0.01 vs. normal diet, Student's t-test), total cholesterol content was almost the same among the three groups (Fig. 2C).

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Fig. 2. Effect of aging on Pai-1 gene expression in the liver of mice fed ad libitum. A: representative electrophoresis photographs of PCR products. B and C: gene expression of Pai-1 (B) and total cholesterol content in the liver (C) of 3-, 6-, and 12-mo-old mice (n = 3 for each group). ##P < 0.01 vs. 3-mo-old mice (one-way ANOVA followed by Fisher PLSD test); **P < 0.01 vs. the normal diet group (two-way ANOVA followed by Student's t-test).
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Recovery from hypercholesterolemia-induced Pai-1 gene expression in the liver.
Two weeks after suspension of the cholesterol diet (4 wk), the enhanced expression (F3,8 = 18, P < 0.01, one-way ANOVA) of Pai-1 in the liver returned to the normal level of expression (F3,8 = 1.6, P > 0.05, one-way ANOVA) (Fig. 3B). There was a significant difference in Pai-1 gene expression (ZT x diet interaction) between normal and cholesterol diet mice (F3,16 = 13.5, P < 0.01, two-way ANOVA). Per2 gene expression in the liver was unaffected by cholesterol intake as evaluated by two-way ANOVA (ZT x diet interaction) (F3,16 = 0.5, P > 0.05).

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Fig. 3. Gene expression of Pai-1 and Per2 in the liver after replacing the cholesterol diet with a normal diet. Mice were fed a cholesterol diet ad libitum for 4 wk, and one-half of the mice (n = 3 for each group) from each group were then fed a cholesterol diet for the next 2 wk, whereas the other one-half were administered a normal diet for the next 2 wk. A: representative electrophoresis photographs of PCR products. The top open and solid bars represent light and dark periods, respectively. B: daily expression of the Pai-1 gene (left) and Per2 gene (right) plotted as the relative mRNA level. **P < 0.01 and *P < 0.05 vs. normal diet group (two-way ANOVA followed by Student's t-test).
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Daily expression of clock and clock-controlled genes in the liver under hypercholesterolemic conditions.
Bmal1, which encodes positive components of the molecular circadian clock (Fig. 4,A and B), peaked at ZT1 or ZT7. Per2, which encodes negative components, peaked at ZT19 (Fig. 4, A and B). Clock-controlled genes such as Dbp and E4bp4 exhibited clear daily rhythms that peaked at ZT13 and ZT1, respectively (Fig. 4B). Hypercholesterolemia did not affect the amplitude or phase of daily rhythms of clock and clock-controlled genes in the liver (Fig. 4B).
Two-way ANOVA revealed significant differences in the daily expression pattern of Pai-1 (ZT x diet interaction) (F3,16 = 12.9, P < 0.01) and Nr4a1 (F3,16 = 11.2, P < 0.01) between normal and cholesterol diet groups. Thus hypercholesterolemia seemed to augment the daily expression of Pai-1 and Nr4a1 mRNA. In addition, mean values for the entire day were 3.9 and 4.5 times higher for Pai-1 and Nr4a1 mRNA levels, respectively, in the cholesterol diet group compared with the normal diet group. On the other hand, there were weak daily rhythms of Tnf-
and Vldlr under both normal and cholesterol diet conditions. However, a cholesterol diet significantly enhanced the induction of these genes as evaluated by one-way ANOVA (main effect of diet) (F1,22 = 5.0, P < 0.05 for Tnf-
mRNA; F1,22 = 10.9, P < 0.01 for Vldlr mRNA).
We found a clear positive correlation between Pai-1 and Nr4a1 gene expression (Fig. 4C; r = 0.84, P < 0.01), a weak correlation between Pai-1 and Tnf-
(r = 0.51, P < 0.05), and no correlation between Pai-1 and Vldlr (r = 0.3, P > 0.05). There were no interactions between Nr4a1 and Tnf-
or Vldlr and Tnf-
and Vldlr .
Effect of SCN lesion on hypercholesterolemia-induced daily expression of the Pai-1 gene.
Four weeks after completion of the cholesterol diet, mice were subjected to lesion of the SCN. Pai-1 and Per2 gene expression after cholesterol intake could be observed 2 wk after SCN lesion. No significant daily rhythms were found in Pai-1 (F3,8 = 0.1, P > 0.05, one-way ANOVA) and Per2 (F3,8 = 1.5, P > 0.05, one-way ANOVA) genes in the liver after SCN lesion (Fig. 5).

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Fig. 5. Gene expression of Pai-1 and Per2 in the liver after suprachiasmatic nucleus (SCN) lesion. Mice fed a cholesterol diet ad libitum for 4 wk were subjected to SCN lesion. SCN-lesioned mice were maintained on a cholesterol diet under ad libitum conditions for another 2 wk to check for behavioral arrythmicity. Intact mice comprised the control groups. Three animals were used for each time point in both the control and SCN-lesioned groups. A: representative electrophoresis photographs of PCR products. The top open and solid bars represent light and dark periods, respectively. B: daily expression of the Pai-1 gene (left) and Per2 gene (right) plotted as the relative mRNA level. **P < 0.01 vs. the control group (two-way ANOVA followed by Student's t-test).
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RF-induced entrainment of clock genes and Pai-1 gene expression.
To exclude the possibility that RF treatment itself affects Pai-1 expression pattern such as level and phase, RF was implemented either during the day or at night in mice previously fed a cholesterol diet under free feeding conditions. As shown in Fig. 6,14 days of daytime RF led to a daily rhythm in Pai-1 gene expression with an advanced peak of expression (ZT7) compared with ad libitum feeding (ZT191). Two-way ANOVA revealed significant differences (ZT x diet interaction) of Pai-1 (F3,16 = 13.6, P < 0.01), Per2 (F3,16 = 16, P < 0.01), and Nr4a1 (F3,16 = 9.3, P < 0.01) expression in the liver of normal and cholesterol diet groups, suggesting the difference in daily expression profile of these genes between normal and cholesterol diet groups. In fact, compared with RF at night, the phase of the peak and trough of these genes was advanced by 612 h with daytime RF.
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DISCUSSION
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In the present experiments, we found a clear daily oscillation of Pai-1 gene expression in the heart and slight expression in the liver of mice fed a normal diet, whereas clock genes such as Per2 and Bmal1 exhibited a clear daily rhythm in both the liver and heart (data not shown). This result is well supported by the findings of Mohri et al. (16), who demonstrated clock-regulated Pai-1 gene expression in the heart but not in the liver under normal diet conditions. In our study, hypercholesterolemia strongly enhanced the daily rhythm of Pai-1 gene expression in the liver but not in the heart. Cholesterol intake upregulated Pai-1 gene expression in the liver but not in the heart, suggesting that the hypercholesterolemia-induced production of PAI-1 may differ in each individual tissue. Such a tissue-specific induction of Pai-1 mRNA was previously reported by Yamamoto et al. (30), who found that Pai-1 mRNA levels in the tissues of restraint-stressed mice had a greater induction in the liver, kidneys, and adrenals and no induction in the lungs, heart, and brain.
The daily oscillation of Pai-1 and Per2 mRNA in the liver of mice fed a cholesterol diet disappeared in the SCN-lesioned mouse liver, suggesting that Pai-1 works as a clock-controlled gene in the liver. Promoter analysis of the Pai-1 gene indicates that the CLOCK-BMAL1 and CLOCK-BMAL2 heterodimer E-box motifs on the Pai-1 gene upregulate PAI-1 synthesis (12, 22). However, hypercholesterolemia did not affect the expression amplitude and phase of clock genes Per2 and Bmal1 and clock-controlled genes Dbp and E4bp4 in the liver. The SCN-lesioned mice exhibited a high expression of Pai-1 in the liver under cholesterol diet, although the rhythmicity was lost. Therefore, the robustness of the daily rhythm of Pai-1 gene expression in the liver with cholesterol intake may not require high expression but only the normal rhythmic expression of clock or clock-controlled genes. Thus a cholesterol diet may not have affected the peripheral clock mechanism in the mouse liver.
In the present experiment, a cholesterol diet significantly facilitated the mean expression level of Tnf-
and Vldlr mRNA throughout the entire day, although the expression of both genes exhibited only a weak rhythmicity. Both TNF-
and VLDL have been shown to increase the biosynthesis of PAI-1 in the endothelial cells and HepG2 cells by inducing transcription of the Pai-1 gene promoter (6, 19, 21, 24). In humans, a positive correlation has been established between elevated very-low-density lipoprotein (VLDL)-triglyceride concentrations and PAI-1 (2). VLDL and VLDL remnants induced by a cholesterol diet are taken up by LDL and VLDL receptors (11, 27). In this experiment, we found a significant increase in hepatic triglycerides after a 6-wk cholesterol diet (3.4 ± 0.4 mg/g tissue for control diet and 5.0 ± 0.3 for cholesterol diet, P < 0.05, Student's t-test). Therefore, VLDL may be responsible for activation of the signaling cascade leading to PAI-1 biosynthesis in the liver at any time of day.
Gruber et al. (8) identified that NR4A1 is an inducible DNA-binding protein that binds specifically to the human Pai-1 promoter in human umbilical vein endothelial cells and that NR4A1 itself is transcriptionally upregulated by TNF-
. When we examined the expression pattern of Tnf-
, Nr4a1, and Vldlr genes in the liver under hypercholesterolemia conditions, we found a strongly associated gene expression between Pai-1 and Nr4a1, a weakly associated expression between Pai-1 and Tnf-
, and no association between Pai-1 and Vldlr. The present results suggest that daily expression of the Pai-1 gene may be strongly controlled by NR4A1, weakly controlled by TNF-
, and not controlled at all by VLDL. Unfortunately, we could not find the NR4A1 response element (AAAGGTCA) in regions of the mouse Pai-1 gene using 10-kbp upstream analyses. Therefore, hypercholesterolemia might independently augment Pai-1 and Nr4a1 gene expression through the activation of TNF-
in the mouse liver. Consequently, the robustness of the daily rhythm of Pai-1 gene expression under hypercholesterolemic conditions may be caused directly and/or indirectly by promoter factors such as NR4A1 and TNF-
and unidentified factors. Under normal diet conditions, promoter regulation on the Pai-1 gene by clock gene-controlled outputs must be masked in the liver and hypercholesterolemia unmasks such an inhibitory regulation. Further experiment is required to find such mask and unmask mechanisms.
In the present experiment, a cholesterol diet produced much greater induction of Pai-1 in the liver of middle-aged mice versus younger mice. In addition, there were higher Pai-1 mRNA levels in the middle-aged mice fed a normal diet compared with the younger mice. Yamamoto et al. (30) demonstrated that 12- and 24-mo-old mice exhibited higher Pai-1 mRNA expression in the liver compared with 8-wk-old mice, and restraint stress produced much greater induction of Pai-1 mRNA in the liver of 12- and 24-mo-old mice. Therefore, although a detailed mechanism of the aging effect on biosynthesis of PAI-1 remains unknown, the results from investigations up to this point suggest that aging, hypercholesterolemia, and stress may all together contribute to the increased risk for thrombosis because of the induction of PAI-1.
We clearly demonstrated through our present results that RF during the daytime caused phase shifts in the daily rhythm of not only Pai-1, Nr4a1, and Tnf-
but also Per2 gene expression in the liver. Therefore, we suggest that peripheral clock genes and clock-controlled gene expression may be associated with the generation of an RF-dependent rhythm. Interestingly, nighttime RF of a normal diet led to a significant daily rhythm of Pai-1 gene expression in the liver, whereas ad libitum feeding (
80% of food intake occurs at night) did not produce a daily rhythm in this study. Therefore, RF during the nighttime may enhance the time cue for RF-induced rhythm oscillation, and we might be able to regulate the timing of fibrinolytic activity through Pai-1 gene expression by changing the feeding schedule.
In summary, a robust day-night rhythm of Pai-1 mRNA expression was observed in the liver of mice fed a cholesterol diet, without affecting clock gene expression. Because a daily change of Pai-1 expression may be implicated in the daily rhythmicity seen with myocardial infarction, the greater risk of acute atherothrombotic events in the morning is augmented under hypercholesterolemic conditions.
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GRANTS
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This study was partially supported by Japanese Ministry of Education, Sports, and Culture Grants 14657621 and 15390074 (to S. Shibata), the Special Coordination Funds of the Japanese Science and Technology Agency.
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ACKNOWLEDGMENTS
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Present address of M. Akiyama: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Science Bldg. 3, Room 214, 2-11-16 Yayoi, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Shibata, Dept. of Pharmacology, School of Science and Engineering, Waseda Univ., Higashifushimi 2-7-5, Nishitokyo-Shi, Tokyo 202-0021, Japan (E-mail: shibatas{at}waseda.jp)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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