Norio Ishida (Prime Senior Researcher of the Institute for Biological Resources and Functions) and Katsutaka Ooishi (Senior Research Scientist) of the Clock Cell Biology Research Group, the Institute for Biological Resources and Functions (Director: Masahiro Iwakura) of the National Institute of Advanced Industrial Science and Technology (AIST) (President: Hiroyuki Yoshikawa), in collaboration with Prof. Hiroyoshi Sei (Faculty of Medicine, the University of Tokushima), found that fibrate, which is an well-known hypolipidemic drug for humans, influences animals' biological seasonal clocks, including hibernation.

By recent changes to 24-hour life and aging society and the rapid progress of information technology, the incidence of seasonal diseases such as winter depression has increased.  For example, the incidence of these syndromes has increased in care facility staff and residents.  Patients with these seasonal diseases show thermoregulatory and sleep features that resemble those of hibernators.  This suggests that non-hibernating mammals such as humans may still have the molecular mechanism associated with hibernation.  Accordingly, elucidation of this mechanism could help us to develop new treatment methods for difficult-to-treat diseases such as winter depression.

This study revealed that mice fed on a diet containing fibrate showed physiological patterns similar to those in the hibernating state.  It is well known that fibrate is a ligand for Peroxisome Proliferator-Activated Receptor α PPARα, a nuclear receptor on liver cells.  In 2007, we found that fibrate advanced the pase of the circadian clock forward and had a therapeutic effect on sleep rhythm disorders called delayed sleep phase syndrome.  In this news , we showed that PPARα effects not only the circadian clock but also the biological seasonal clock.

Parts of the results will be published in the October 2008 issue of Endocrinology (published by the Endocrine Society).  The results will be exhibited as "New Drugs target from Studies of Biological Clocks" at the AIST Open Lab held at the AIST Tsukuba on October 20 and 21, 2008.

There have recently been recent progress in the study of the molecular mechanism of the biological clock in mammals.  It has long been known that animals have a seasonal rhythm.  Observational studies have showed that the hibernation rhythm is altered in bears and squirrels whose the brain master circadian clock are lesioned, suggesting the relationship between the circadian clock and the seasonal clock.  However, no reports have elucidated the relationship between them in terms of the molecular mechanism.

The number of patients with sleep rhythm disorders has increased in Japan along with phenomena such as aging and 24-hour society.  Furthermore, there has been an increase in the incidence of circadian rhythm sleep disorders and winter depression; these are recognized as social problems associated with a high frequent use and dependense of information technology.  Recent data (Survey of Potential Depression, 11 April 2008, Pfizer Japan Inc.) have shown that 1/8 of ordinary peoples  and 1/3 of staffs of the care facilities for aged persons suffered depression.  Therefore, it is becoming an important project to understand seasonal clocks at a molecular level.

Gene-level study of AIST has revealed that the fatty acids degradation system is strongly regulated by biological clocks.  In particular, in the liver, we showed that the rhythmic expression of the nuclear receptor PPARα, which plays a key role in fatty acid degradation, is controlled by a biological clock in 2005.  In 2007,  Mr.Hidenori Shirai, Clock Cell Biology Group in AIST found that fibrate, a lipid-lowering drug that works on PPARα, had an "early bird effect" on model mice that could not rise in the morning (delayed sleep-phase syndrome): in other words, the drug advanced time of the circadian clock.  The results were announced to the press on 25th April, 2007. After this discovery, AIST has been performing a study on the effect of fibrate on the biological seasonal clock.

The effect of fibrate administration to advance the circadian clock was revealed in the experiments under the even day/night condition (12 hours day; 12 hours night).  To elucidate the mechanism of the mouse seasonal clock, mice were housed under the conditions in an artificially longer daytime (long-day like summer; 18 hours day, 6 hours night) or artificially longer nighttime (short day like winter; 6 hours day, 18 hours night).  Intersestingly, a phase-advance effect of fibrate administration on mice locomotor was observed only in the long-day mice, but not in the short day (Figure 1).

Fig. 1 Representative circadian behavioral rhythms of mice administered with bezafibrate under different photoperiods. (a-d) Mice were given bezafibrate under light-dark (LD) 16:8 (a,b) or LD 8:16 (c,d), then returned to a normal diet and transferred to constant darkness. Left ordinate indicates days after bezafibrate administration. Dark phase duration is shaded in grey. Horizontal open and solid bars, day and night, respectively.

Next, 24-hour body temperature, electroencephalogram, and electromyogram were measured in mice fed for 2 weeks on a diet containing fibrate and kept under 12-hour day and 12-hour night conditions.  Interstingly enough, Fibrate caused low body temperature(daily torpor) and deep NREM sleep similar to hibernation in laboratory mouse.  After sleep deprivation, both of the deep NREM sleep time and the total sleep time were increased.  Deep NREM sleep was continuously high in the fibrate administration group at 6 hours of sleep deprivation.  Figure 2 shows the body temperature (1) before and (2) after administration of fibrate and (3) standard diet for 5 weeks after fibrate administration.  The results indicate that the low body temperature-effects of fibrate disappeared after feeding the standard diet for 5 weeks.

Fig. 2 The effect of bezafibrate on body temperature (BT). (A) Time course (above) of BT in mice fed a control diet (open circles), a bezafibrate-supplemented diet (filled circles) or the control diet following recovery (gray circles).

In order to elucidate the molecular mechanism how fibrate influences the biological seasonal clock in mice body, we carried out a study aimed at screenig which substances increases in the brain to fibrate administration.  The results revealed that the mRNA contents of neuropeptide Y (NPY) increased in the hypothalamus (Figure 3).  NPY is considered to play an important role in the promotion of eating and obesity and energy adjustment; this remind us a close relationship between lipid metabolism and the biological seasonal clock. We also found that liver FGF21 was significantly increased at the same timing of low body temperature.

FGF21 is a novel endocrine hormone which regulate lipid and glucose metabolism.

Fig.3 Real-time RT-PCR analysis of gene expression in the hypothalamus at ZT10 and ZT22. Expression of Npy and 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 versus control (Mann-Whitney U-test). Data represent means ± SEM (n = 4 per group).

These phenomena are characteristic of hibernators, suggesting that the molecular mechanism involved in hibernation remains in non-hibernating mammals such as humans and laboratory mice.  This is the first data that the circadian clock drives our internal seasonal clock through the nuclear receptor PPARα-FGF21-NPY pathway.  Further studies of this molecular mechanism will promote the development of new treatment methods or drugs for difficult-to-treat diseases such as winter depression.

We aim to test antidepressant effects and anxiolytic effects in mice by administering fibrate; we also want to identify the transmitter from the peripheral clock to the brain clock. The molecular mechanism advancing the biological clock under long-day conditions is our next goal.  We hope that the results of bezafibrate to molecular clock mechanisms will provide key information for the development of drugs for difficult-to-treat diseases such as winter depression and overeaters.