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Biological Clock in the Liver

Hitoshi Okamura

33

33.1

Introduction

Most organisms living on earth have an internal clock and thus circadian rhythm represents a ba- sic feature of life. The discovery of the mammalian clock genes and their oscillatory mechanisms have brought the research on biological clocks to a vari- ety of fields of life science and medicine. In many organisms, the circadian core oscillator is thought to be composed of an autoregulatory transcription- (post)translation-based feedback loop involving a set of clock genes [19]. In mammals, the discovery of the clock genes and their oscillation in most of the cells in the body, including hepatocytes, changed the concept of circadian oscillatory system in mam- mals. Now, the feature of the circadian system is the prevalence of the oscillation at the level of genes, re- flected at cell, tissue, and system levels. In this chap- ter, I shall briefly summarize the basic system of cir- cadian rhythms in mammals, then describe how the molecular clocks in hepatocytes regulate the timing of regeneration of liver clocks, and finally explain the adjustment of liver clocks to their environmen- tal time cues.

33.2

Circadian Oscillatory System in Mammals The principal oscillator of circadian rhythms is lo- cated in the suprachiasmatic nucleus (SCN) of the hypothalamus [28]. Since the destruction of this nu- cleus induces the arrhythmicity of locomotor activ- ity and hormonal rhythms, this oscillator is thought to be the master oscillator of most of the circadian rhythms in the body such as behavioral rhythms (e.g., sleep–wake cycle) and hormonal rhythms (e.g., cortisol and melatonin).

However, the special status of the SCN as the cir- cadian oscillator was threatened by the recent find- ings that peripheral organs have the clock genes and

their molecular oscillating ability; at least in certain conditions, peripheral tissue can oscillate (thus we will call this the peripheral clock). The concept that most peripheral cells are oscillating has been report- ed in Drosophila [20] and zebra fish [56], in which the peripheral clocks can be entrained directly by light [48, 57]. In the liver, which hosts a powerful pe- ripheral oscillator, the circadian clock is entrained by a restriction of feeding [17], and this entrainment is independent from the SCN [23]. In a fibroblast cell line, external stimuli such as a high concentra- tion of serum and endothelin can induce the circa- dian expression of the clock genes for several cycles [6, 7, 59]. Molecular oscillatory components and their oscillatory mechanism of central clocks (in the SCN) and peripheral clocks (represented by fi- broblasts) are mostly identical [59]. However, tissue

Fig. 33.1. Circadian system in mammals spanning the gene, cell and system levels, featuring the SCN central clock. Gene depicts rhythmic transcription of mPer1 and mPer2. P and N at gene level represent positive and negative elements, respectively. Cell rep- resents neuronal electrical activities of single SCN neurons. Sys- tem indicates the sum of the local neuronal and glial circuits of the SCN

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PART II: Specifi c Signaling Pathways

culture studies have demonstrated that SCN clocks oscillate for more than a month , although clocks in peripheral tissues slow down after a few cycles [62]. In the SCN, multi-phased thousands of clock oscillating cells synchronize and produce a stable and robust rhythm [61], which is transmitted to the peripheral tissues. Since destruction of the SCN abolishes rhythms in clock gene expression in the liver [43], the signals from SCN are crucial not only for entrainment, but also for sustaining the oscilla- tion of biological clocks in peripheral tissues. Thus, it is now thought that the mammalian clock system displays a complex hierarchical structure headed by the eternally oscillating SCN at the top (Fig. 33.1).

33.3

Cellular Core Feedback Loop of Clock Genes The molecular feedback loops generating circadian oscillation in each cell clock can be summarized as follows. The oscillation of each cell clock starts first at the transcription of two main oscillators mPer1 and mPer2 [5, 46, 63]. The heterodimers formed by the bHLH-PAS proteins (CLOCK and BMAL1) bind to the E-box of mPer1 and mPer2 promoters [31],

and initiate the transcription of these mPer genes [22]. Activated transcription results in the forma- tion of mPer1 and mPer2 mRNAs, which are trans- lated in the cytoplasm to mPER1 and mPER2 pro- teins. These proteins translocate into the nucleus, and form negative complexes that comprise mCRY1, mCRY2, mPER1, mPER2, mPER3 and mTIM, and that suppress the transcription of the mPer1 and mPer2 genes by binding to the positive factors (CLOCK/BMAL1) (Fig. 33.2). Since mCry1/mCry2 double knockout mice and Bmal1 (Mop3) knock- out mice [11] show the immediate loss of behavioral rhythm in constant darkness, mCry1/mCry2, and Bmal1 play a key role in making up the core loop.

Phosphorylation of mPER1 and mPER2 by ca- sein kinase I ε (CKIε) is the first evidence that pro- tein level regulation is crucial for determining the circadian period length [32, 54]. Furthermore, there is growing evidence that clock proteins are regu- lated dynamically in both spatial (nuclear and cy- toplasmic) and temporal (production and degrada- tion) dimensions (Fig. 33.2). The main clock oscil- latory protein mPER2 usually shuttles between the cytoplasm and the nucleus and is easily degraded by ubiquitination and the proteasome pathway [60].

Recently, it has been shown that ubiquitination and proteasome-dependent degradation of mPER pro-

Fig. 33.2. Model of the core feedback loop in the mammalian circadian clock. BMAL1/CLOCK heterodimer binds to E-box in clock oscillating mPer1 and mPer2 genes to accelerate their transcription. The core negative autoregulatory feedback loop is regulated at protein level by a negative complex consisting of mPER1, mPER2, mPER3, mCRY1 and mCRY2. mPER2 protein

is produced in the cytoplasm, phosphorylated by casein kinase 1ε(CK1ε). mPER2 protein keeps on shuttling between nucleus and cytoplasm via the CRM1/Exportin1 nuclear export system until (a) mPER2 is ubiquitinated and subsequently degraded by the proteasome system or (b) the stabilization of nuclear mPER2 by the binding of mCRY1 or mCRY2. See text for details

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teins occur in mammalian cells [2, 60]. It is also evident that the ubiquitination of mPER proteins is inhibited in the presence of mCRY proteins and the mPER proteins appear to be more fragile if they do not dimerize with mCRY proteins. Since mCRY protein, which is the strongest suppressor of mPer1 transcription, can be ubiquitinated when mPER proteins are absent [60], the stabilized negative complex suppresses mPer1 and mPer2 transcription, and shuts off the clock. Re-starting the clock gene transcription depends on the nuclear export ability of mPER proteins [54, 60]. The decrease of mPER in the nucleus by the CRM1/Exportin1 nuclear export machinery causes destabilization of mCRY, and the decrease of mCRY will lead to the beginning of mPer1 and mPer2 gene transcription.

33.4

Expression Profiles of Clock Genes and Their Outputs in the Liver

What are the expression profiles of clock genes in the liver? Liver mPer1 mRNA showed a peak at CT12 (where CT0 is subjective dawn and CT12 is subjec- tive dusk) and a trough at CT0 [7, 33]. mPer2 mRNA begins slightly after mPer1 mRNA, showing a peak at CT16 and a trough at CT0–4. The phase difference of mPer1 and mPer2 mRNA found in liver was also observed in the SCN [50, 51, 64]. More interestingly, the expression profiles of mPer1 and mPer2 in the liver are phase-advanced for 4–8 h compared with those of the SCN (Fig. 33.3a). At the protein level, mPER2 protein appeared at CT20 in the liver, which is 4–8 h before it appeared in the SCN. Although the reasons for the phase difference between central and peripheral clocks are not known, this difference sug- gests that hourly steps exist to initiate the peripheral clock oscillation, or the SCN-entraining factors are released at its decreasing time (long after the peak time) in clock gene expression. For other core clock composing genes, BMAL1 mRNA, a positive factor for mPer1 and mPer2 gene transcription, showed inverted expression profiles to mPer1 and mPer2 mRNA, and clock mRNA is constantly expressed all day long in the liver, as in the SCN (Fig. 33.3a).

The core oscillatory loop composed of clock genes is thought to be common in all cell clocks re- gardless of SCN and peripheral organs. However, the outputs from this oscillatory loop are different and unique to a specific cell type. Gene array studies have demonstrated that hundreds of genes are con- trolled by the circadian clock [3, 39] with its tissue specificity. In the liver, many enzyme-coding genes show circadian characteristics [3, 29, 39] . Many of

these genes, which have a similar peak–trough ex- pression profile to mPer genes, are regulated by two routes. The first is E-box (CACGTG, CACGTT)-me- diated mechanisms directly controlled by CLOCK:

BMAL1 heterodimers. This is observed in many genes and its representative is the vasopressin gene regulation [26]. The second is an indirect pathway of D-box (RTTAYGTAAY: R, purine; Y, pyrimi- dine), consisting of antagonistic regulation of PAR proteins and E4BP4 [35], which is also used as the accessory feedback loop of mPer genes. Albumin, cholesterol 7 α hydroxylase and cytochrome P450 (Cyp2A5) and possibly aromatic l-amino acid de- carboxylase are regulated by the D-box mechanism [25, 30], in which the positive PAR proteins and the negative E4BP4 switch back and forth between the on–off conditions of the target genes. In addition, RevErba/ROR responsive elements (AGGTCA) are known to be important for making night-time peak rhythms [41].

33.5

Circadian Clock and Liver Regeneration

Since the life span of each cell is limited, cell growth and mitosis should be performed continuously to retain the organ or tissue. There is substantial evi- dence that the circadian rhythms affect the timing of cell divisions in vivo. Day–night variations in both the mitotic index and DNA synthesis were found in oral mucosa [8], intestinal/rectal epithelium [10, 44], corneal epithelium [13], and bone marrow [47].

Since some of them are shown to persist in constant darkness [13, 44], these rhythms may be under the control of an endogenous rhythm. These studies were performed in normal physiological conditions by histochemical techniques, but the mitotic cells were at most only a small proportion of the non-mi- totic cells. To reveal further the molecular mecha- nisms between regeneration and the circadian clock, we must search for a more suitable system in which biochemical techniques can be applied.

The liver is an interesting organ since a vigorous process of regeneration follows partial removal of liver tissue [24, 34], although the cell cycle period is extremely long in the unoperated condition. A two thirds partial hepatectomy (PH) induced the large majority of the pre-existing hepatocytes into the regenerative process, undergoing one or more cell division cycles [4, 34, 36]; the regeneration speed is rapid and the liver mass restored within 7 days [14, 18, 24, 34].

Applying PH to mice, Matsuo et al. [33] analyzed

the molecular connection between the circadian

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PART II: Specifi c Signaling Pathways

Fig. 33.3a–e. Expression of clock genes in liver and effect of partial hepatectomy (PH) on liver regeneration. a Schematic rep- resentation of the circadian expression profiles of mPer1, mPer2, mClock and mBMAL1 mRNA in the liver. b, c Time course of the expression of BrdU-stained hepatocytes (b) and mitotic hepato- cytes (c) after PH at Zeitgeber time (ZT)8 and ZT0. Values in the graphs show mean percentages±SEM. d, e Liver regeneration in Cry-deficient mice. d Time course of mitosis in Cry-deficient mice after PH. e Liver weights of wild-type (PH/ZT8 and PH/ZT0) and Cry-deficient (PH/ZT8) mice 72 h and 10 days after PH. Liver weight before the operation was adjusted to 100%. Values rep- resent the mean±SEM. (n=4–6; *p<0.01 and **p<0.05, respec- tively). —(b, e From [33])

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clock and the cell cycle. After PH, most of the hepa- tocytes entered the cell cycle, and restored the liver mass within a few days. The rate of liver re-growth was studied in mice housed in a 12-h light–12-h dark cycle, with a PH on the liver performed at ZT8 or ZT0 (ZT, Zeitgeber time; ZT0 is the start of the light period, and ZT12 is the start of the dark period). S- phase kinetics represented by the incorporation of bromodeoxyuridine (BrdU) were similar in both the ZT0 and the ZT8 operation (Fig. 33.3b). However, there was an 8-h delay in cells entering mitosis (M- phase) when PH was performed at ZT0 (Fig. 33.3c), compared with PH at ZT8. This indicates the tim- ing of the hepatectomy is crucial for the entry to M- phase in these regenerating cells.

To elucidate the molecular mechanism underly- ing the difference of the entry of M-phase of the cell cycle, DNA arrays and Northern blots were applied.

Eleven out of 68 cell-cycle-related genes showed a difference in PH/ZT8 and PH/ZT0, with three genes (cyclin B1, cdc2, and wee1) showing a remarkable difference [33]. Cyclin B1 and cdc2 were positively correlated, and wee1, a known Cdc2 regulator, were negatively correlated to M-phase. This is interesting since all these genes are cell cycle regulators govern- ing G

2

–M transition, and these expression profiles correlate well to the M-phase progress.

To elucidate how cell cycles work in animals with- out molecular clocks, the PH was applied to Cry1

–/–

Cry2

–/–

(Cry-deficient) mice in which all clocks in both central and peripheral oscillators were stopped [38]. In these mice, the mitosis (Fig. 33.3d) and Cdc2 kinase activity were severely impaired, and the liver regeneration was severely blunted, at least in the initial phase (Fig. 33.3e). To address the mechanism of blunted cell cycle in Cry-deficient mice, we com- pared the expression profiles of the cell-cycle-relat- ed genes in Cry-deficient mice with those in wild- type mice. Genes showing a difference in expression were the M-phase-related cyclin B1, cdc2, wee1, Bub1 and p55CDC, the S-phase- and M-phase-related cy- clin A2, and the G

1

-phase-related cyclin D1. Among these, the expression profiles of cyclin D1 and wee1 transcripts showed a marked difference throughout the regeneration process.

The above studies indicate that expression pro- files of the wee1 gene correlate with the mitotic progress in the circadian phase. In normal mice liver, there were clear circadian rhythms of wee1 expres- sion with very low levels at CT0/CT4 and high levels at CT8/CT12/CT16 (Fig. 33.4a). Levels of wee1 were increased in Cry-deficient mice, whereas they were severely suppressed in Clock mutant (Clock/Clock) mice, which carry dominant negative Clock muta- tions. These expression profiles resembled those of clock-controlled genes via E-box such as dbp.

Indeed, in the 5’ UPR of wee1, there were three E- boxes (CACGTG), which were activated by CLOCK/

BMAL1 and suppressed by PER2, PER3, CRY1 and CRY2 (Fig. 33.4b, c). When these E-box regions were mutated, transcription of wee1 by CLOCK/BMAL1 was decreased. This suggests that the wee1 tran- scription is directly regulated by the core feedback loop through the E-box elements.

Does the transcriptional change of the wee1 gene reflect at protein level, and finally influence cdc2 activity levels? WEE1 protein expression and WEE1 kinase activity were well correlated to wee1 mRNA expression, in which they were all negatively correlated with mitotic peak [33]. The kinetics of phosphorylation of Cdc2 on Tyr15, which is pre- dominantly carried out by the WEE1 kinase [55], was indeed coupled to WEE1 kinase activity. In Cry- deficient mice, wee1 mRNA, WEE1 protein levels, WEE1 kinase activity and p-Cdc2(Tyr15) were very high after PH, which accounts for the low activity of the Cdc2 kinase in Cry-deficient mice.

The above finding strongly suggests that the cel- lular molecular loop of the circadian clock directly regulates WEE1, a kinase that inhibits mitosis by inactivating Cdc2/cyclin B (Fig. 33.4d). External humoral and/or neural timing cues participating in cell cycle regulation may use this cellular circadian oscillatory loop. Recently, another example suggest- ing the association of circadian and cell growth has also been demonstrated, where arrhythmic mice lacking the PER2 protein are more sensitive to ra- diation and develop spontaneous lymphomas with a much higher frequency than wild-type siblings [21]. Possibly, the inactivation of mPer2 could lead to derepression of Myc expression, resulting in un- controlled cell growth and tumor formation.

As the cell cycle has autonomous activity, there

may be another fundamental clock, which is differ-

ent from the circadian clock: the cell cycle clock con-

trols cell division, and the circadian clock controls

various clock-controlled genes [15]. Although clas-

sically thought to be independent, a recent demon-

stration of derepression of Myc in mPer2 knockout

mice, and activation of wee1 by clock genes, chal-

lenges this view. Unfortunately, very little is known

about the association of circadian rhythms and tu-

mor growth. However, it is clear that these studies

contribute innovative insights towards the problems

of chronomedicine: timing of massive hepatectomy

or application of anticancer drugs.

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PART II: Specifi c Signaling Pathways

Fig. 33.4a–d. Circadian regulation of the wee1 gene. a Circadian fluctuation of wee1 transcripts in wild-type mice. Note the con- stant increase in Cry-deficient mice and decrease in Clock/Clock mice. b Location of the E-box sites within the 5’-flanking region of the mouse wee1 gene. The arrow indicates the transcription start site. c Transcriptional regulation of the mouse wee1 gene by clock genes. Reporter plasmid containing the 1.2-kb mouse

wee1 5’-upstream region, including the three E-boxes (wee1 1.2 kb) or mutated E-boxes (all three E-boxes were mutated to 5’-CT- GCAG-3’; mutant 1.2 kb), was used for the transcriptional assay.

Presence (+) or absence (–) of the reporter and expression plas- mids is shown. Each value represents the mean±SEM. d Sche- matic representation showing the link of circadian clock and cell cycle. See text for further details. (a–c From [33])

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33.6

Glucocorticoids as a Serum Resetting Factor In previous paragraphs, I have explained the mo- lecular mechanism of oscillation of the cell clock in liver and its outputs featuring the signal linking the molecular clock and the cell cycle. How do these cell clocks know the timing of being awake? Also, are SCN signals crucial for the local oscillation of peripheral clocks? In mammals, light information processed in the retina is converted to an electrical signal in ganglion cells, which directly projects to the hypothalamic SCN and adjusts the SCN clock to the environmental light–dark cycles. The adjusted SCN pacemaker produces standard time (Fig. 33.5), and these signals are transmitted to peripheral timekeepers by neuronal and/or humoral signals [28, 49].

Because serum induces circadian gene expres- sion in cultured rat-1 fibroblasts [6], some blood- borne factors must stimulate the molecular oscil- lators in peripheral cells. It has already been dem- onstrated that dexamethasone [7], endothelin [59], forskolin [58] and phorbol ester [1] induce the circa- dian rhythms in fibroblast cell lines. Among these, glucocorticoid hormones are particularly attractive candidates, because they are secreted physiologi- cally in daily cycles [53] and the glucocorticoid re- ceptor (GR) is expressed widely in most peripheral cell tissues [42]. To prove that glucocorticoids are a humoral factor mediating SCN and peripheral clocks, Balsalobre et al. [7] injected the glucocorti- coid hormone analog dexamethasone 21-phosphate (2 mg/kg) with transient change of the phase of cir- cadian gene expression in liver, kidney, and heart.

However, contrary to the distinct window of light- induced phase-response curve (PRC) in the central

Fig. 33.5. Central clocks and peripheral clocks. Molecular oscil- lator is common in both central and peripheral clocks. Central clock in SCN is the only clock that can regulate another clock at

system level. Outputs of SCN regulate behavioral rhythms via non-SCN brain clocks, and peripheral clocks via glucocorticoids and the sympathetic nerve

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PART II: Specifi c Signaling Pathways

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SCN clock [16], the PRC of dexamethasone in liver indicates that the phase of peripheral oscillators can be changed throughout the 24-h day [7] (Fig. 33.6).

Since dexamethasone failed to induce mPer1 mRNA expression in mutant mice (GR

AlfpCre

) with a hepa- tocyte-specific inactivation of the GR gene [7, 27],;

with the clear induction of mPer1 mRNA in kidney and heart, a functional GR is necessary for the acti- vation of mPer1 transcription after serum glucocor- ticoid stimulus.

33.7

Noradrenaline as a Neuronal Resetting Factor As well as the above humoral factors, the autonomic nervous system is also a candidate linking the SCN to the periphery [9, 45], and it is suspected to play a fundamental role in the circadian homeostasis of sleep–wake cycles, cardiovascular, respiratory, and gastrointestinal functions [12]. These circadian–au- tonomic interactions have been characterized in the view of light information processing, which is

Fig. 33.6a–d. Dexamethasone induces phase shifts in circa- dian gene expression in the periphery but not in the SCN. a Circadian Per1 expression in liver after dexamethasone (Dex, 400 µg/ml) injection. b The levels of DBP and Rev-erbα mRNAs were determined by ribonuclease protection experiments. c The signals obtained in ribonuclease protection assays for DBP and TBP transcripts. The relative levels of DBP transcripts are given as DBP/TBP mRNA signal ratios on the y axis. Dotted lines indi-

Fig. 33.7. Visualization of elevated mPer1 transcription by electrical stimulation (EST) of the sympathetic nerve.

The splanchnic nerve he- patic branch was electrically stimulated for 30 min (3 Hz, 5 V, 5 ms). Upper left: lumines- cence before stimulation; up- per right: luminescence after stimulation. Luminescence was recorded by a two-di- mensional photon-counting camera. Luminescence in the liver was increased without affecting the front and rear paws. Images displayed with pseudocolor represent ac- cumulated photo counts. The site of EST is shown in detail in the lower panel. (Adapted from [52])

cate peak expression; differences indicate phase shift. d Dex- amethasone-induced phase shifts in the expression of DBP and Rev-erbα transcripts were recorded after injections at Zeitgeber time (ZT)1, ZT3, ZT5, ZT8, ZT10, ZT11, ZT14, ZT18, ZT21, and ZT23.

Open circles DBP mRNA, filled circles Rev-erbα mRNA. For esthet- ic reasons, the values obtained at ZT5 and ZT8 are shown at the beginning and the end of the phase-response curve. (Adapted from [7])

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known as the most well-characterized and strongest time cue of the circadian rhythm [40]. Evidence is accumulating that the light signal integrated in the SCN is transmitted to peripheral organs via central and peripheral autonomic nuclei and nerves [9, 37].

Photic stimulus modulates the efferent activities of sympathetic (adrenal, hepatic, and pancreatic branches of the splanchnic nerve) and parasympa- thetic (hepatic and pancreatic branches of the vagus) nerves in rats, and lesions of the anterior hypotha- lamus abolish the responsiveness [37]. The ACTH- independent acute suppression of corticosterone by light is speculated to be transmitted via the auto- nomic innervation to the adrenal cortex [9].

Terazono et al. [52] analyzed the clock gene ex- pression in the liver with pharmacological appli- cation of various adrenergic drugs in vitro and in vivo. They demonstrated that the administration of adrenaline or noradrenaline increased the expres- sion of mPer1 but not mPer2 in the liver under in vivo conditions and in vitro hepatic slices. Electrical stimulation of the sympathetic nerves or adrenaline injection caused an elevation of bioluminescence in the liver of transgenic mice carrying the mPer1 promoter luciferase (Fig. 33.7). In the liver, mPer1 and mPer2 mRNA showed peaks at subjective dusk to early night, with the trough at subjective dawn to early morning. After chemical denervation of no- radrenergic nerves by 6-hydroxydopamine pretreat-

Fig. 33.8a, b. Liver rhythms after the chemical denervation of noradrenergic fibers and SCN lesion. a Degeneration of sympa- thetic nerve by 6-OHDA abolishes/diminishes mPer1 and mPer2 rhythms in the liver. Each animal was injected with either saline (open circles, three animals per point) or 6-OHDA (closed circles, four or five animals per point) at Zeitgeber time (ZT)12 on days 1 and 3. On day 4, animals were killed at each ZT. Relative mRNA level of mPer1 or mPer2 is shown as the ratio to β-actin mRNA level. b Effect of daily adrenaline injection on mPer1, mPer2, and mBmal1 gene expression in the liver of SCN-lesioned, arrhythmic

mice. Shown are images after electrophoresis (right graph) and relative mRNA levels of mPer1, mPer2, mBmal1, and β-actin RT- PCR products obtained from intact mice (open circles, three to six animals per point), daily saline-injected SCN-lesioned mice (open triangles, three to six animals per point), and daily adrena- line-injected SCN-lesioned mice (closed circles, three to five ani- mals per point). Relative mRNA level of mPer1, mPer2, or mBmal1 is shown as the ratio to β-actin mRNA level. Dotted lines repre- sent duplicate data at ZT3, 5, 7, and 23 in the respective groups.

(Adapted from [52])

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ment (6-OHDA), circadian rhythms of liver mPer1 and mPer2 mRNA were blunted or disappeared (Fig. 33.8a). Lesion of the SCN also completely abol- ished the mPer1 and mPer2 mRNA rhythms. Inter- estingly, however, after daily injection of adrena- line at ZT22 (2 h before dawn), mPer1 and mPer2 rhythms recovered in the liver even in SCN-lesioned animals (Fig. 33.8b). This indicates that the pulsatile daily adrenergic stimulus can induce the rhythms in the liver, and suggests that adrenergic signals may be the mediator linking the central SCN clock to pe- ripheral clocks.

The unique feature of circadian biology is that gene transcription almost perfectly reflects the be- havioral and physiological rhythms. This means that the clock gene oscillation generated by the core loop in each SCN neuron is coupled and am- plified, and spread into the whole brain and to all the peripheral organs including liver through oscil- lation conducting systems, where glucocorticoids and sympathetic nerves play important roles. In the liver, clock signals entrain the cell clocks, and the intracellular oscillating molecular loop coordinates the timing of the expression of a variety of genes with specific cellular function. Whether regulation is humoral or neural is interesting when analyzing the circadian expression of the variety of genes af- ter liver transplantation, although the association of liver transplantation and the circadian regulation of hepatic functions is poorly understood. If humoral factors such as glucocorticoids are crucial, rhyth- mic expression of clock genes might occur just after the liver transplantation, but if adrenergic nerves have a dominant role, the circadian expression will occur later, after the re-innervation of nerves to the donor liver. In addition, after the hepatectomy, the timing of expression of the cell cycle gene wee1 is circadian-regulated. “Time” governs liver functions as a whole.

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PART III

Horizons III

Chapter 34

Pharmacogenomics of Cholestatic Liver Disease 407

Christiane Pauli-Magnus, Marie V. St.-Pierre, Peter J. Meier Chapter 35

Proteomics of Signal Transduction Pathways 417

Oliver Kleiner, Jasminka Godovac-Zimmermann

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