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Genes to Cells (2004) 9, 857-864. doi:10.1111/j.1365-2443.2004.00769.x
© 2004 Blackwell Publishing or its licensors
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Effect of feeding on peripheral circadian rhythms and behaviour in mammals

Hisato Kobayashi1,{dagger}, Katsutaka Oishi1,{dagger}, Shuji Hanai1 and Norio Ishida1,2,*

1 Clock Cell Biology, Institute of Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
2 Institutes of Applied Biochemistry, University of Tsukuba, Ibaraki 305-8502, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Although feeding time is a dominant cue for circadian rhythms in mammalian peripheral tissue, the effect of feeding and fasting on circadian gene expression and behaviour is unknown. Here we report that fasting does not affect the phase of rhythmic mRNA expression levels of the clock genes, mPer1, mPer2 and of the clock controlled gene, mDBP. However, the levels of each of these genes were significantly altered in different ways and recovered by feeding. We also found that feeding enhances phase-shifting to a new light-dark cycle of rhythmic mPer2 mRNA expression in the heart. Furthermore, feeding enhances the phase-shifting to new light-dark cycle of behaviour more than fasting. Our data indicate that feeding is an important cue for circadian behaviour rhythms as well as for the photo-entrainment of peripheral clock gene expression.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus is the central oscillator that controls the approximately 24-h periodicity (circadian rhythms) of mammalian behaviour and physiology (Dunlap 1999; Ishida et al. 1999; Schibler & Sassone-Corsi 2002). Neurones of the SCN receive light information via the retinohypothalamic tract, and the phase of circadian clock is adapted to the photoperiod (Dunlap 1999; Hastings 1997; Takahashi 1995). Meanwhile, not only the SCN but also other tissues and cells are equipped with endogenous oscillators. In fact, expression of the mammalian period (per) genes that are homologues of the Drosophila clock gene, is robustly circadian not only in the SCN but also in other peripheral tissues (Dunlap 1999; Ishida et al. 1999; Oishi et al. 1998; Sakamoto et al. 1998; Schibler & Sassone-Corsi 2002; van Esseveldt et al. 2000). Feeding has recently been considered as a dominant Zeitgeber (timing cue) to peripheral clocks, except for the central clock in the SCN. Daytime feeding of nocturnal mice and rats completely inverts the phase of circadian gene expression in peripheral tissues, but does not affect the central clock (SCN) (Damiola et al. 2000; Stokkan et al. 2001; Schibler & Sassone-Corsi 2002). However, the effects of fasting on circadian gene expression and of feeding on the shift to a new LD cycle of mice behaviour remain unknown. Thus, we investigated the effects of fasting and feeding on circadian gene expression and behaviour of mice.

The present study found that fasting affects the RNA expression levels of mouse circadian genes but does not alter the peak times of each individual gene. We also found that feeding enhances phase-shifting to a new light-dark cycle (LD cycle) of mPer2 mRNA expression in the heart and circadian behaviour. Thus, feeding is an important cue for the photo-entrainment of peripheral clock gene expression and of circadian behaviour.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Fasting altered the level of clock gene mRNA

To determine whether fasting affects the circadian system in peripheral tissues, we examined circadian gene expression in mice that were provided with food ad libitum (ad libitum feeding) or not (fasting). We then determined the mRNA expression profiles of mPer1, mPer2, and mDBP (Albumin D-site binding protein) in the livers and hearts of the fasting mice (Fig. 1). The expression profiles of clock genes have been characterized as convenient markers that indicate the phase of the circadian clock for each organ (Dunlap 1999), and the mRNA expression profiles are similar in both tissues (Storch et al. 2002). Here, we maintained Jcl:ICR mice under a 12 : 12 dark-light cycle (DL cycle) for over 2 weeks. Fasting followed the start of the dark term. Mice were sacrificed at specific times (Zeitgeber time 14, 17, 20, 23, 2, 5, 8 and 11; ZT12–24: dark term, ZT0–12: light term) for 3 days under fasting conditions. A comparison of the expression profiles in fed and fasting mice showed that fasting induces tissue- and gene-specific alterations of mRNA expression. Under fasting conditions, the expression level of mPer1 was increased and that of mDBP was reduced in rat heart and liver. The expression level of mPer2was reduced in the liver, but not altered in the heart under fasting. On day 3 after fasting, the peak level of mPer1 mRNA showed 2.0 times, 2.5 times higher before fasting in heart and liver, respectively, whereas that of mPer2 mRNA was reduced half before fasting in rat liver. The peak level of mDBP was reduced to about 40% before fasting in both tissues. The data indicate that fasting affected significantly the mRNA expression levels of these circadian genes in peripheral tissues by unknown mechanism. In contrast, the peak times of these genes expression around dusk (ZT11–14) was the same in mice fed ad libitum, suggesting that the fasting does not affect the phase of peripheral circadian rhythms.



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  		Figure 1  Fasting influences circadian clock gene expression in the heart and liver. Left, heart; right, liver (A) Representative Northern blots of mPer1, mPer2, mDBP and GAPDH. Open and solid bars represent lights-on and off, respectively. (B) mRNA levels of each gene. Maximal gene expression under ad libitum feeding is expressed as 100%.

 
Altered mRNA levels of clock genes are recovered by feeding

To determine whether the changes induced by fasting were reversible, we examined clock gene expression in mice that resumed feeding after fasting (REF group). Mice were fed at ZT0 after a 36-h fast (36 h-REF group) or at ZT12 after a 48-h fast (48 h-REF group). The 36-REF mice were sacrificed at ZT2, 5, 8, 11, 14, 17, 20 and 23, and the 48-REF mice at ZT14, 17, 20, 23, 2, 5, 8 and 11 (Fig. 2). We compared the results after resuming feeding with those obtained under either conditions of fasting or ad libitum feeding. These results demonstrated that the mRNA levels of each gene recovered to those of mice fed ad libitum in both tissues. However, re-feeding did not affect the peak times of these genes (around ZT11–14), although the level of mPer2 mRNA in the liver was 8-fold increased at ZT5 (5 h after re-feeding) in the 36 h-REF group.



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  		Figure 2  Fasting-induced changes in heart and liver circadian gene expression returned to the levels under ad libitum feeding, and re-feeding induced liver mPer2 mRNA. Left, heart; right, liver (A) Representative Northern blots analysis of mPer1, mPer2, mDBP and GAPDH. Open and solid bars represent lights on and off, respectively. (B) Quantification of clock gene mRNA in fasting (closed circle), 36 h-REF (open diamonds), 48 h-REF (open triangles), and ad libitum (open circles) groups. Maximal gene expression under ad libitum feeding is expressed as 100%.

 
Feeding ad libitum enhances the light-induced phase-shift of circadian gene expression in peripheral tissues to a new LD cycle

To examine whether feeding is required to shift the phase of peripheral clocks, we examined mPer2 expression in mice that were fed ad libitum (RevAD group) or fasted (RevFA group) under a reversed LD cycle (Fig. 3). We determined the mRNA expression profiles of mPer2 in the hearts of the mice, because fasting does not change these profiles. We also compared the findings with those of mice under a non-reversed LD cycle (ad-lib and fasting groups). We determined the mRNA expression profiles of mPer2 in the hearts of mice. Jcl:ICR mice were maintained under a 12 : 12 DL cycle for over 2 weeks, and then this cycle was reversed at the end of the light term (Time 0). Mice were sacrificed at eight time points per day for 3 days (Time 2–71), and then their hearts were dissected. At day 1 of the reversed LD cycle, the peak time of mPer2 mRNA expression was advanced about 4-h in the RevAD and RevFA groups. At day 3, the peak time of mPer2 mRNA expression was advanced a further 8-h in the RevAD, but not in the RevFA group. These data showed that ad libitum feeding enhances the light-induced phase-shift of mPer2 mRNA expression in peripheral tissues to a new LD cycle more than fasting.



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  		Figure 3  Feeding enhanced phase-shifted mPer2 mRNA expression in the heart more than fasting. (A) Representative Northern blot of mPer2. Open and solid bars represent lights on and off, respectively. (B) Quantification of mRNA in ad-lib (open circles), RevAD (closed circles), fasting (open diamonds), and RevFA (closed diamonds) groups. Maximal mPer2 mRNA level under ad libitum feeding is expressed as 100%. Significant differences between values from two groups are shown as *P < 0.01 at each time point.

 
Entrained behaviour to new LD cycle under fasting was delayed compared with that under ad libitum feeding conditions

To examine whether fasting affects the phase of behavioural circadian rhythms, we measured the frequency of drinking activity and compared the phase shift of new LD condition from ad libitum and fasting mice (Fig. 4A). A representative of drinking activity showed that entrained behaviour to new LD cycle under fasting was delayed compared with that under ad libitum feeding conditions. To quantify such results, we analysed the frequency of drinking activity in the RevAD and RevFA groups (Fig. 4B). The frequency of drinking activity was concentrated during the dark term in the ad-lib and fasting groups at day 3. In contrast, the shift to new LD cycle were detected in the RevAD group, but the mice remained active during the light term in the RevFA group at day 3. These data showed that behaviour entrained to a new LD cycle under the fasting condition was significantly delayed compared with that under ad libitum feeding conditions. Thus, feeding seems to be important for the entrainment of behaviour and of peripheral clock gene expression to a new LD cycle.



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  		Figure 4  Change in behaviour rhythm to a new LD cycle attenuated under fasting. (A) Representative double-plotted actogram of drinking activity in ad-lib, RevAD, fasting, and RevFA groups. Lighting for RevAD and RevFA groups was changed from DL (Time 0–12: dark term, Time 12–24: light term) to LD cycles (Time 0–12: light term, Time 12–24: dark term). (B) Graph shows rates of drinking activity in each group. White and black bars show light and dark term activities, respectively. Total daily activity is expressed as 100%. All values are expressed as means ± SEM (n = 3–4). Significant differences between values of two groups are shown as *P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Although fasting altered the level of clock gene mRNA, the length of periodicity was not affected (Fig. 1). Our data also showed that neither fasting nor resumed feeding changes the phase of peripheral clocks under LD cycles. Among the genes examined, only the level of mPer2 mRNA expression in the heart was not affected by fasting, thus we selected mPer2 expression in the heart as a marker which could indicate the phase of peripheral clocks even under conditions of fasting. We found that the mPer2 mRNA level was rapidly induced at ZT5 in the liver of the 36 h-REF group (Fig. 2). During that period, the acute induction of mPer2 mRNA seems to play a role in the phase shifting of peripheral clocks. The mPer2 expression that is induced by resumed feeding might be a initial event in the inverted phase of mPer2 mRNA expression in the liver of nocturnal rodents under restricted feeding during the light period (Damiola et al. 2000; Stokkan et al. 2001).

We showed here that feeding is important for the entrainment of peripheral circadian gene expression and behaviour rhythms to new LD cycles (Figs 3 and 4). Photo-entrainment seems to arise via a fast step advance during day 1 and a slow step on day 3. The former can slightly phase-shift mPer2 expression even during fasting. The latter is food dependent and seems to be critical for the phase reversal of peripheral clocks for restricted feeding. These data indicate that food is a very important cue for the entrainment of peripheral circadian rhythms to a reversed LD cycle.

Food-dependent synchronization might involve a cellular redox state like an NAD balance (Schibler & Sassone-Corsi 2002). Experiments in vitro have shown that the binding activity of NPAS2 and BMAL1 (or CLOCK), which are components of the molecular feedback loop of circadian rhythms, depends on the rate of reduced and oxidized NAD cofactors (Reick et al. 2001; Rutter et al. 2001). Furthermore, glucose affects rPer1 and rPer2 mRNA expression in Rat-1 fibroblasts (Hirota et al. 2002). Such a metabolic pathway might cause the phase-shift of mPer2 mRNA expression induced by feeding during the reversed LD cycle. Furthermore, we found that fasting differentially affected the expression of circadian genes in the heart and liver. Expression of the mPer1, mPer2 and mDBP genes, which have E-box elements in their promoter region, is considered for CLOCK/BMAL dependent transcription in vivo (Gekakis et al. 1998; Ripperger et al. 2000; Yamaguchi et al. 2000). Gene-specific changes of these mRNA levels by fasting suggest transcriptional regulation, which is not simply explained by E-box dependent regulation. Moreover, fasting might affect the mRNA expression of some metabolic genes that are included in circadian gene expression in the liver. In fact, the expression of some metabolic genes is circadian in the liver, but not in the heart (Storch et al. 2002). Further studies should examine the effects of metabolic processes on circadian gene expression.

The effects of peripheral circadian clocks on circadian behaviour and physiology remain unknown. However, we found that both phases of peripheral clock gene expression and of the frequency of drinking activity could not completely entrain mice that had fasted for 3 days to a new LD cycle. One possible explanation is that parasympathetic and sympathetic control from peripheries like pancreas might affect the function of SCN by food intake (Bujis et al. 2001). The circadian system in the SCN and in other areas of the brain under the same conditions might reveal a relationship among these clocks, peripheral circadian clocks and circadian behaviour. Understanding the role of food in peripheral clocks and circadian behaviour will bring new insights into mammalian physiology and behaviour.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Animals

Male 8- to 12-week-old Jcl:ICR mice (Clea JAPAN. Tokyo) were maintained under a 12 : 12-h LD cycle at room temperature (25 ± 0.1 °C) and given food and water ad libitum before the experiment. A white fluorescent lamp was the light source during the day (200–300 lux at cage level).

RNA Isolation and northern blotting

Mice (n = 3–4 for each time point) were killed at specific times. Excised hearts and livers were rapidly isolated, frozen in liquid nitrogen and stored at –80 °C. Total RNA isolated from frozen specimens using Isogen (guanidine HCl/phenol procedure; Nippon Gene, Tokyo, Japan), was separated on 1% agarose/0.7 M formaldehyde gels and then blotted on to nylon membranes (GeneScreen Plus; DuPont, USA) by passive capillary transfer. Lanes contained 20 mg of total RNA from each tissue. 32P-labelled probes were generated from the cDNA fragments of mPer1 (bases: 2358–3117; GENBANK Accession No. AB002108), mPer2 (bases: 1123– 1830; GENBANK Accession No. AF036893), mDBP (bases: 1138–1602; GENBANK Accession No. J03179), and samples were normalized by measuring the amount of GAPDH mRNA (bases: 149–571; GENBANK Accession No. M32599).

Analysis of drinking activity

Throughout the experiment, mice were housed in aluminium-net cages. Drinking activity was continuously recorded using Chronobiology Kits (Stanford Software Systems, Stanford, CA, USA). Drinking rhythms were measured by infrared sensors placed beside the nozzle of the water bottle. The results are displayed as relative average activity counts during the light and dark terms.

Data analysis

All values are expressed as means ± SEM (n = 3–4). One-factor ANOVA followed by the Bonferroni/Dunn test statistically analysed the data.


   Footnotes
 
Communicated by: Shu Narumiya

{dagger}These two authors contributed equally to this work. Back

* Correspondence: E-mail: n.ishida{at}aist.go.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Bujis, R.M., Chun, S.J., Niijima, A., Romijin, H.J., et al. (2001) Parasympathetic and sympathetic control of the pancreas: a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. J. Comp Neurol. 431, 405–423.[CrossRef][Medline]

Damiola, F., Le Minh, N., Preitner, N., et al. (2000) Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 14, 2950–2961.[Abstract/Free Full Text]

Dunlap, J.C. (1999) Molecular bases for circadian clocks. Cell. 96, 271–290.[CrossRef][Medline]

van Esseveldt, K.E., Lehman, M.N. & Boer, G.J. (2000) The suprachiasmatic nucleus and the circadian time-keeping system revisited. Brain Res. Rev. 33, 34–77.[CrossRef][Medline]

Gekakis, N., Staknis, D., Nguyen, H.B., et al. (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569.[Abstract/Free Full Text]

Hastings, M.H. (1997) Circadian clocks. Curr. Biol. 7, R670–R672.[CrossRef][Medline]

Hirota, T., Okano, T., Kokame, K., et al. (2002) Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured Rat-1 fibroblasts. J. Biol. Chem. 277, 44244–44251.[Abstract/Free Full Text]

Ishida, N., Kaneko, M. & Allada, R. (1999) Biological clocks. Proc. Natl. Acad. Sci. USA 96, 8819–8820.[Abstract/Free Full Text]

Oishi, K., Sakamoto, K., Okada, T., et al. (1998) Humoral signals mediate the circadian expression of rat period homologue (rPer2) mRNA in peripheral tissues. Neurosci. Lett. 256, 117–119.[CrossRef][Medline]

Reick, M., Garcia, J.A., Dudley, C., et al. (2001) NPAS2: an analog of clock operative in the mammalian forebrain. Science 293, 506–509.[Abstract/Free Full Text]

Ripperger, J.A., Shearman, L.P., Reppert, S.M., et al. (2000) CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev. 14, 679–689.[Abstract/Free Full Text]

Rutter, J., Reick, M., Wu, L.C., et al. (2001) Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors. Science 293, 510–514.[Abstract/Free Full Text]

Sakamoto, K., Nagase, T., Fukui, H., et al. (1998) Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J. Biol. Chem. 273, 27039–27042.[Abstract/Free Full Text]

Schibler, U. & Sassone-Corsi, P. (2002) A web of circadian pacemakers. Cell 111, 919–922.[CrossRef][Medline]

Stokkan, K.A., Yamazaki, S., Tei, H., et al. (2001) Entrainment of the circadian clock in the liver by feeding. Science 291, 490–493.[Abstract/Free Full Text]

Storch, K.F., Lipan, O., Leykin, I., et al. (2002) Extensive and divergent circadian gene expression in liver and heart. Nature 417, 78–83.[CrossRef][Medline]

Takahashi, J.S. (1995) Molecular neurobiology and genetics of circadian rhythms in mammals. Annu. Rev. Neurosci. 18, 531–553.[CrossRef][Medline]

Yamaguchi, S., Mitsui, S., Miyake, S., et al. (2000) The 5’ upstream region of mPer1 gene contains two promoters and is responsible for circadian oscillation. Curr. Biol. 10, 873–876.[CrossRef][Medline]

Received: 13 April 2004
Accepted: 28 June 2004





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