FEBS Letters 584 (2010) 1597–1601

journal homepage: www.FEBSLetters .org

Altered circadian rhythm of the clock genes in fibrotic livers inducedby carbon tetrachloride

Peng Chen a, Xiamusiya Kakan a,b, Jianfa Zhang a,*

a Center for Molecular Metabolism, Nanjing University of Science and Technology, Nanjing 210094, Chinab Department of Agriculture, Xinjiang Agricultural University, Urumchi 830052, China

a r t i c l e i n f o

Article history:Received 25 February 2010Revised 12 March 2010Accepted 12 March 2010Available online 15 March 2010

Edited by Veli-Pekka Lehto

Keywords:Liver fibrosisCircadian clock geneCosine methodMice

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.03.019

Abbreviations: CCl4, carbon tetrachloride; HSC, hsmooth muscle alpha-actin; Bmal1, brain and musperiod; Cry, cryptochrome; SCN, suprachiasmatic nuclperoxisome proliferator-activated receptor alphaoxidoreductase

* Corresponding author.E-mail address: jfzhang@mail.njust.edu.cn (J. Zhan

a b s t r a c t

Disruption in circadian rhythms either by mutation in mice or by shiftwork in people, is associatedwith an increased risk for the development of multiple organ diseases. In turn, organ disease mayinfluence the function of clock genes and peripheral circadian systems. Here we showed that hepaticfibrosis induced by carbon tetrachloride in mice leads to alterations in the circadian rhythms ofhepatic clock genes. Especially, we found an impaired daily Cry2 rhythm in the fibrotic livers, withmarkedly decreased levels during the day time while compared with control livers. Associatively,the expressions of two important clock-regulated genes peroxisome proliferator-activated receptoralpha and cytochrome P450 oxidoreductase lost circadian rhythm with significantly decreased lev-els during the light–dark (12/12 h) cycle in fibrotic livers.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Circadian rhythms are daily cycles of physiology and behaviourwhich are driven by an endogenous oscillator with a period ofapproximately one day [1–3]. A broad spectrum of physiologicalparameters including the sleep–wake cycle, hormone secretion,heart beat, blood flow and body temperature fluctuate with a per-iod of 24 h [4]. In mammals, the master circadian clock is located inthe suprachiasmatic nuclei (SCN) [5], and the peripheral circadianoscillator also exists in several organs including liver. Disruption incircadian rhythms either by mutation in mice or by shiftwork inpeople, is associated with an increased risk for the developmentof multiple organ diseases [6,7]. In turn, a lot of pathological pro-cesses such as Alzheimer’s disease, obesity/type-2 diabetes andhypertension influence the circadian expressions of clock genesin peripheral tissue [8–10].

As the final common pathway of most chronic liver diseases[11], liver fibrosis which leads eventually to cirrhosis and hepato-cellular carcinoma is the major causes of morbidity and mortality

chemical Societies. Published by E

epatic stellate cell; a-SMA,cle Arnt-like protein 1; Per,ei; ZT, zeitgeber time; PPARa,; POR, cytochrome P450

g).

worldwide [12,13]. It is reported that a variety of circadian param-eters, including fibribolysis [14], variceal bleeding [15], heptaticcatabolism of melatonin and plasma melatonin [16,17], arterialblood pressure and heart rate [18,19], gastric acidity [20] and evensleep [21,22] are disrupted in cirrhosis patients or mice. In view ofthe reality that altered circadian rhythms of physiological parame-ters are observed in cirrhosis patients, it is of interest to explore therelationship between liver fibrogenesis and hepatic circadian clockgenes expression. In the present study, we compared the expres-sions of hepatic circadian clock genes and two important clock-regulated hepatic metabolic genes, peroxisome proliferator-acti-vated receptor alpha (PPARa) and cytochrome P450 oxidoreductase(POR), to explore the impact of fibrogenesis on hepatic circadiansystem.

2. Materials and methods

2.1. Animals

Male 7–8 week old C57BL/6 mice were used in this work.Animals were maintained in 12/12 h light/dark cycles with lighton at 7:00 am and off at 7:00 pm, and with free access to reg-ular chow food and water. All animal care and use procedureswere in accordance with the guidelines of the Institutional Ani-mal Care and Use Committee at Nanjing University of Scienceand Technology.

lsevier B.V. All rights reserved.

Table 1Primer sequences used for real-time PCR.

Gene Forward/Reverse

Primer (50–30)

Gapdh ForwardReverse

CATCCACTGGTGCTGCC AAGGCTGT

ACAACCTGGTCCTCAGTGTAGCCCA

Bmal1 ForwardReverse

ACATAGGACACCTCGCAGAA

AACCATCGACTTCGTAGCGT

Clock ForwardReverse

CGGCGAGAACTTGGCATT

AGGAGTTGGGCTGTGATCA

Per1 ForwardReverse

TCCTCAACCGCTTCAGAGATC

CGGGAACGCTTTGCTTTAGA

Per2 ForwardReverse

GTGAAGCAGGTGAAGGCTAAT

AAGCTTGTAAGGGGTGGTGTAG

Cry1 ForwardReverse

TTGCCTGTTTCCTGACTCGT

GACAGCCACATCCAACTTCC

Cry2 ForwardReverse

TCGGCTCAACATTGAACGAA

GGGCCACTGGATAGTGCTCT

PPARa ForwardReverse

GGGTACCACTACGGAGTTCACG

CAGACAGGCACTTGTGAAAACG

POR ForwardReverse

GAAGAGCTACGAGAACCAG

TCAGGTACAGCTCCTTGC

1598 P. Chen et al. / FEBS Letters 584 (2010) 1597–1601

2.2. Experimental model of fibrosis

Hepatic fibrosis was induced by intraperitoneal injection of0.6 ml/kg of body weight of carbon tetrachloride (CCl4) whichwas diluted in olive oil twice-a-week for 4 weeks and controlgroup received the same volume of vehicle [23]. One day afterthe last injection mice (without fasting) were sacrificed at zeitge-

Fig. 1. Representative images of Masson’s trichome staining and a-SMA immunohistocholive oil and CCl4. The collagen deposition showed in blue was markedly observed in fibimmunohistochemistry, in control mice, very weak positive response exists (C) while i�100.

ber time1 (ZT1) (ZT0 corresponds lights on and ZT12 to light off),ZT5, ZT9, ZT13, ZT17, ZT21 (n = 5–6, each time point).

2.3. Histological analyses and immunohistochemistry

A small piece of liver tissue was collected and fixed in 10% buf-fered formalin. The sample was then embedded in paraffin, slicedinto 5-lm-thick sections. H&E and Masson’s trichrome stainingwere performed to monitor lipid accumulation and collagen depo-sition, respectively. For smooth muscle alpha-actin (a-SMA) detec-tion, immunohistochemical analysis was described previously [24].Briefly, the deparaffinized liver sections were treated with 5% non-fat milk to inactivate endogenous peroxidase, and incubated withprimary antibody (a-SMA) at 4 �C overnight. Then slides werewashed and incubated at room temperature with biotinylated goatanti-rabbit antibody. Final incubation was carried out for 1 h withan avidin–horseradish peroxidase complex, and positive reactionswere visualized with DAB.

2.4. Quantitative real-time PCR

Total RNA from liver sample was extracted using Trizol (Invitro-gen, CA) according to the manufacturer’s instruction. Reverse tran-script reaction was carried out by KeyGene reverse transcriptenzyme according to the manufacturer’s protocol. The real-timePCR reaction was carried out on ABI 7300 real-time PCR systemwith cDNA sample and amplified in a 20 ll reaction volume con-taining 1� SYBR Green PCR master mix (Applied Biosystems, CA).The primers used in this work were shown in Table 1. Relative

emistry of the livers from control and fibrosis mice after twice-a-week injection ofrosis group (B) whereas in control mice, little collagen was detected (A). For a-SMAn fibrosis mice, markedly HSC activation was observed (D). Original magnification

P. Chen et al. / FEBS Letters 584 (2010) 1597–1601 1599

expression in comparison with Gapdh was calculated by the com-parative CT method.

2.5. Data analysis and statistics

Results were expressed as mean ± S.E.M. The single cosinormethod was used for analysis of circadian rhythm [10,25], andthe cosine function equation was as follows: Y (t) = M +Acos(xt + u). The rhythm characteristics estimated by this methodincluded the mesor (middle value of the fitted cosine representinga rhythm-adjusted mean), the amplitude (half the differencebetween the minimum and maximum of the fitted cosine func-tion), and the acrophase (time of peak value in the fitted cosinefunction). A probability value determined from comparison ofresiduals, before and after cosine curve fitting, of 60.05 indicateddetection of a rhythm. Rhythm characteristics (mesor, amplitude,and acrophase) were compared between groups by unpaired Stu-dent t test and differences were considered significant at P < 0.05.

3. Results

3.1. Hepatic fibrosis and hepatic stellate cell activation were observedafter chronic CCl4 treatment

To confirm hepatic fibrosis was induced in CCl4 treated mice,Masson’s trichome staining was performed to show the hepaticcollagen deposition. As shown in Fig. 1A and B, the control mice in-jected with oil only displayed limited collagen staining whereas inCCl4 group, mice developed fibrous septa around the centrilobularand formed bridging fibrosis. Because activated hepatic stellate cell(HSC) was the main source of collagen, and a-SMA expression wasthe key marker of HSC activation [11], we monitored the HSC acti-

0 4 8 12 16 20 24

0.0

0.3

0.6

0.9

Rel

ativ

e Bm

al1

mR

NA

leve

l

Zeitgeber Time

M = 0.30

0 4 8 12 16 20 24

0.0

0.3

0.6

0.9

Rel

ativ

e Bm

al1

mR

NA

leve

l

Zeitgeber Time

M = 0.31 M = 0.30

0 4 8 12 16 20 24

0.3

0.6

0.9

1.2

Rel

ativ

e C

lock

mR

NA

leve

l

Zeitgeber Time

M = 0.54

0 4 8 12 16 20 24

0.3

0.6

0.9

1.2

Rel

ativ

e C

lock

mR

NA

leve

l

Zeitgeber Time

M = 0.76

0 4 8 12 16 20 240

6

12

18

24

30

Rel

ativ

e Pe

r1 m

RN

A le

vel

Zeitgeber Time

M = 5.36

0 4 8 12 16 20 240

6

12

18

24

30

Rel

ativ

e Pe

r1 m

RN

A le

vel

Zeitgeber Time

M = 12.78

Rel

ativ

e Pe

r2 m

RN

A le

vel

0

0

1

1

Rel

ativ

e C

ry1

mR

NA

leve

l R

elat

ive

Cry

2 m

RN

A le

vel

lortnoC Fibrosis

Fig. 2. Circadian expression of clock genes in control and fibrosis mice. Levels of mRexpressions of Bmal1 and Per1 were attenuated and the mesors in the expressions of Clowere significantly delayed. Circadian rhythm of Cry2 expression was lost in fibrosis grou

vation using a-SMA immunohistochemistry in control and CCl4

group. As shown in Fig. 1C and D, in control group, a-SMA stainingwas restricted to vessel walls while in CCl4 group a-SMA-positivearea was observed around the centrolobular and infiltrated the lob-ule. These results demonstrated that liver fibrosis was successfullyestablished in CCl4 treated group.

3.2. Altered hepatic clock genes expression in liver fibrosis mice

The core components of clockwork consists of a transcriptionalfeedback loop in which clock and brain and muscle Arnt-like pro-tein 1 (Bmal1) play the positive regulation role while period(Per) and cryptochrome (Cry) act as the negative regulators [1,2],thus it mainly involves in six members, Bmal1, Clock, Per1, Per2,Cry1 and Cry2 [4]. Normally, most of these genes exhibit circadianexpression pattern in both primary circadian oscillator (SCN) andperipheral tissues including liver [3]. To investigated possible ef-fects of hepatic fibrogenesis on clock genes expression, we ana-lyzed the hepatic diurnal expression of transcripts encodingCLOCK, BMAL1, PER1, PER2, CRY1 and CRY2 in the control andfibrosis mice at ZT1, ZT5, ZT9, ZT13, ZT17, ZT21 (ZT0 correspondslights on and ZT12 to light off). As shown in Fig. 2, statistical anal-ysis with the single cosinor method revealed that highly significant24-h variations in the expression of almost all clock genes exam-ined were observed in control and fibrosis mice. Table 2 showedthe mesors of Clock and Per1 were significantly higher in fibrosismice. The amplitudes of the variations in Bmal1 and Per1 were sig-nificantly lower in fibrosis group. Additionally, the acrophases ofClock, Per1 and Cry1 were delayed in fibrosis mice compared withcontrol mice. Especially, the rhythmic expression of Cry2 was to-tally disrupted with dramatically reduced during day time (ZT1,ZT5 and ZT9) in fibrosis animals. Notably, there were no significant

0 4 8 12 16 20 24

0 4 8 12 16 20 24

0 4 8 12 16 20 240 4 8 12 16 20 24

0

10

20

30

40

Zeitgeber Time

M = 12.64

0 4 8 12 16 20 240

10

20

30

40

Rel

ativ

e Pe

r2 m

RN

A le

vel

Zeitgeber Time

M = 12.72

.4

.8

.2

.6

Zeitgeber Time

M = 0.70

0 4 8 12 16 20 24

0.4

0.8

1.2

1.6

Rel

ativ

e C

ry1

mR

NA

leve

l

Zeitgeber Time

M = 0.71

0.2

0.4

0.6

0.8

1.0

1.2

Zeitgeber Time

M = 0.69

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e C

ry2

mR

NA

leve

l

Zeitgeber Time

sisorbiFControl

NA were determined by quantitative real-time PCR. The amplitudes of circadianck and Per1 were increased. Acrophases for the expressions of Clock, Per1 and Cry1p. Data represent means ± S.E.M. (n = 5–6).

Table 2Circadian rhythmic parameters of clock gene transcriptions in control and liverfibrosis mice.

Gene Mesor Amplitude Acrophase ZT (h)

ControlBmal1 0.30 ± 0.03 0.35 ± 0.03 23.15 ± 0.07Clock 0.54 ± 0.06 0.26 ± 0.04 0.52 ± 0.09Per1 5.36 ± 0.56 5.38 ± 0.49 12.23 ± 0.15Per2 12.64 ± 4.25 13.91 ± 4.71 14.06 ± 0.27Cry1 0.70 ± 0.09 0.48 ± 0.05 19.43 ± 0.12Cry2 0.69 ± 0.10 0.24 ± 0.04 6.00 ± 0.49PPARa 1.13 ± 0.23 0.85 ± 0.22 8.90 ± 0.02POR 0.63 ± 0.11 0.24 ± 0.06 12.90 ± 0.39

FibrosisBmal1 0.31 ± 0.05 0.22 ± 0.04* 0.39 ± 0.12Clock 0.76 ± 0.10* 0.19 ± 0.04 4.39 ± 0.32**Per1 12.78 ± 2.29** 3.50 ± 0.43* 14.27 ± 0.62**Per2 12.72 ± 3.60 11.78 ± 4.16 15.18 ± 0.81Cry1 0.71 ± 0.10 0.44 ± 0.05 21.48 ± 0.69**Cry2 – – –PPARa 0.41 ± 0.10* 0.12 ± 0.08* 9.67 ± 2.58POR 0.37 ± 0.07* 0.04 ± 0.002* 11.27 ± 2.46

Data represent means ± S.E.M., *P < 0.05, **P < 0.01 vs. control group.

1600 P. Chen et al. / FEBS Letters 584 (2010) 1597–1601

differences in rhythmic parameters of Per2 in both groups. Collec-tively, these data indicated that the expression of hepatic clockgenes has been altered in the fibrosis mice.

3.3. Attenuated rhythmic mRNA expression of PPARa and POR infibrotic liver

Altered circadian genes expression implied that a series ofdownstream clock-controlled genes would change their expressionrhythm. Then we examined the expressions of PPARa and POR infibrotic liver by quantitative RT-PCR. As shown in Fig. 3, highly sig-nificant circadian rhythms in the expressions of PPARa and PORwere observed in control group, however, in fibrosis animals, themesors of both PPARa and POR were significantly lower and theamplitudes of both genes were markedly attenuated (Table 2).Therefore, these data revealed that in fibrotic liver, the impairedclock genes expression resulted in disrupted clock-regulated genesexpression.

0 4 8 12 16 20 240.0

0.8

1.6

2.4

Zeitgeber Time

Rel

ativ

e PP

AR a

lpha

M = 1.13

0 4 8 12 16 20 240.0

0.8

1.6

2.4

Zeitgeber Time

M = 0.41

0 4 8 12 16 20 24

0.4

0.8

1.2

1.6

Rel

ativ

e PO

R m

RN

A le

vel

Zeitgeber Time

M = 0.63

0 4 8 12 16 20 24

0.4

0.8

1.2

1.6

Rel

ativ

e PO

R m

RN

A le

vel

Zeitgeber Time

M = 0.37

sisorbiFlortnoC

mR

NA

leve

l

Rel

ativ

e PP

AR a

lpha

m

RN

A le

vel

Fig. 3. Hepatic expression of PPARa and POR in control and fibrosis mice. Levels ofmRNA were determined by real-time quantitative PCR. The mesors and amplitudesof the expression of both genes were dramatically attenuated in fibrosis mice. Datarepresent means ± S.E.M. (n = 5).

4. Discussion

In the present study, we compared the liver clock genes expres-sion in control and fibrosis mice to provide direct evidence thatmolecular clockwork was disrupted in fibrotic liver. The amplitudeof circadian expression of clock genes (Clock, Bmal1, Per1, Cry1 andCry2) were significantly changed in fibrotic livers compared withcontrol livers, except for Per2. Martino et al. showed that someclock gene rhythms in cardiac hypertrophic mice could remain un-changed and they suggested that rhythmicity was an importantcontributing mechanism to compensatory hypertrophy [26]. In thiswork, notably, Per2 expression rhythm was not obviously shiftedand altered in fibrotic livers, which may implied that the rhythmicexpression of Per2 gene could be an important contribution mech-anism to liver fibrosis development. In our previous observation,mice deficient in Per2 exhibited markedly sensitive to chronicCCl4 treatment compared with WT mice [27]. Hence, Per2 geneplayed a protective role in chemical-induced liver failure.

With alterations of the rhythmic expression of clock genes in fi-brotic liver, consequently, some clock-controlled genes, such asPPARa and POR, lost their circadian expression rhythm with a se-verely declined mRNA levels. PPARa and POR were both clock-reg-ulated genes and exhibited circadian expression pattern in liver[28,29]; PPARa had been shown involving in many physiologicalprocesses such us lipid metabolism, inflammatory responses, oxi-dative stress and controls of cell cycle [30]. The cytochromeP450s belonged to a superfamily that played a central role in thedetoxification of xenobiotics, as well as in the metabolism ofendogenous compounds such as steroids, fatty acids, prostaglan-dins and leukotrienes [31]. POR was a very important factor in reg-ulating the activity of cytochrome P450s and the downstreamprocesses [31]. Thus, both of them were upstream compoundsand we thought that their expression might reflect the metabolismstatus in liver. It was reported that PPARa deficient mice showeddefect in lipid metabolism with enhanced hepatic lipid accumula-tion [32]. In this work, the expression of PPARa was dramaticallyreduced in fibrotic livers compared with control livers; liver stea-tosis was also correlated with liver fibrosis [33]. POR knockoutmice exhibited a profound decrease in all hepatic microsomalP450 functions and compromised drug metabolism [31]. In addi-tion, Barreiro et al. had provided a piece of evidence in supportof a correlation between liver fibrosis and impaired drug metabo-lism [34]. Therefore, changes of many physiological parameters(e.g. lipid and drug metabolism) in fibrosis animals might be attrib-uted by the impaired PPARa and POR expression.

In conclusion, our observations demonstrate that liver fibrogen-esis change the hepatic circadian expression of clock genes. Furtherstudy is to explore the detail about how liver fibrogenesis affectsthe clock genes expression and how does the altered clock genesexpression affect the circadian physiology in liver cirrhosis.

Acknowledgement

This work was supported by the National Science Foundation ofChina (30730030) a Program for New Century Excellent Talents inUniversity.

References

[1] Hastings, M.H., Reddy, A.B. and Maywood, E.S. (2003) A clockwork web:circadian timing in brain and periphery, in health and disease. Nat. Rev.Neurosci. 4, 649–661.

[2] Rutter, J., Reick, M. and McKnight, S.L. (2002) Metabolism and the control ofcircadian rhythms. Annu. Rev. Biochem. 71, 307–331.

[3] Portman, M.A. (2001) Molecular clock mechanisms and circadian rhythmsintrinsic to the heart. Circ. Res. 89, 1084–1086.

[4] Albrecht, U. and Eichele, G. (2003) The mammalian circadian clock. Curr. Opin.Genet. Dev. 13, 271–277.

P. Chen et al. / FEBS Letters 584 (2010) 1597–1601 1601

[5] Schibler, U. and Sassone-Corsi, P. (2002) A web of circadian pacemakers. Cell111, 919–922.

[6] Kennaway, D.J., Owens, J.A., Voultsios, A., Boden, M.J. and Varcoe, T.J. (2007)Metabolic homeostasis in mice with disrupted clock gene expression inperipheral tissues. Am. J. Physiol. 293, R1528–R1537.

[7] Ha, M. and Park, J. (2005) Shiftwork and metabolic risk factors ofcardiovascular disease. J. Occup. Health 47, 89–95.

[8] Wu, Y.H., Fischer, D.F., Kalsbeek, A., Garidou-Boof, M.L., van der Vliet, J., vanHeijningen, C., Liu, R.Y., Zhou, J.N. and Swaab, D.F. (2006) Pineal clock geneoscillation is disturbed in Alzheimer’s disease, due to functional disconnectionfrom the ‘‘master clock”. FASEB J. 20, E1171–E1180.

[9] Ando, H., Yanagihara, H., Hayashi, Y., Obi, Y., Tsuruoka, S., Takamura, T.,Kaneko, S. and Fujimura, A. (2005) Rhythmic messenger ribonucleic acidexpression of clock genes and adipocytokines in mouse visceral adipose tissue.Endocrinology 146, 5631–5636.

[10] Mohri, T., Emoto, N., Nonaka, H., Fukuya, H., Yagita, K., Okamura, H. andYokoyama, M. (2003) Alterations of circadian expressions of clock genes inDahl salt-sensitive rats fed a high-salt diet. Hypertension 42, 189–194.

[11] Friedman, S.L. (2000) Molecular regulation of hepatic fibrosis, an integratedcellular response to tissue injury. J. Biol. Chem. 275 (4), 2247–2250.

[12] Prosser, C.C., Yen, R.D. and Wu, J. (2006) Molecular therapy for hepatic injuryand fibrosis: where are we? World J. Gastroenterol. 12 (4), 509–515.

[13] Török, N.J. (2008) Recent advances in the pathogenesis and diagnosis of liverfibrosis. J. Gastroenterol. 43, 315–321.

[14] Piscaglia, F., Siringo, S., Hermida, R.C., Legnani, C., Valgimigli, M., Donati, G.,Palareti, G., Gramantieri, L., Gaiani, S., Burroughs, A.K. and Bolondi, L. (2000)Diurnal changes of fibrinolysis in patients with liver cirrhosis and esophagealvarices. Hepatology 31, 349–357.

[15] Mann, N.S., Hillis, A., Mann, S.K., Buerk, C.A. and Prasad, V.M. (1999) Incirrhotic patients variceal bleeding is more frequent in the evening andcorrelates with severity of liver disease. Hepatogastroenterology 46 (25), 391–394.

[16] Steindl, P.E., Ferenci, P. and Marktl, W. (1997) Impaired hepatic catabolism ofmelatonin in cirrhosis. Ann. Intern. Med. 127 (6), 494.

[17] Steindl, P.E., Finn, B., Bendok, B., Rothke, S., Zee, P.C. and Blei, A.T. (1995)Disruption of the diurnal rhythm of plasma melatonin in cirrhosis. Ann. Intern.Med. 123 (4), 274–277.

[18] Møller, S., Wiinberg, N. and Hernriksen, J.H. (1995) Noninvasive 24-hourambulatory arterial blood pressure monitoring in cirrhosis. Hepatology 22 (1),88–95.

[19] Alvarez, D., Golombek, D., Lopez, P., de las Heras, M., Viola, L., Sanchez, S.,Kolker, M. and Mastai, R. (1994) Diurnal fluctuations of portal and systemichemodynamic parameters in patients with cirrhosis. Hepatology 20 (5), 1198–1203.

[20] Savarino, V., Mela, G.S., Zentilin, P., Mansi, C., Mele, M.R., Vigneri, S., Cutela, P.,Vassallo, A., Dallorto, E. and Celle, G. (1996) Evaluation of 24-hour gastricacidity in patients with hepatic cirrhosis. J. Hepatol. 25 (2), 152–157.

[21] Córdoba, J., Cabrera, J., Lataif, L., Penev, P., Zee, P. and Blei, A.T. (1998) Highprevalence of seep disturbance in cirrhosis. Hepatology 27, 339–345.

[22] Montagnese, S., Middleton, B., Mani, A.R., Skene, D.J. and Morgan, M.Y. (2009)Sleep and circadian abnormalities in patients with cirrhosis: features ofdelayed sleep phase syndrome? Metab. Brain Dis. 24 (3), 427–439.

[23] Perugorria, M.J., Latasa, M.U., Nicou, A., Cartagena-Lirola, H., Castillo, J., Goñi,S., Vespasiani-Gentilucci, U., Zagami, M.G., Lotersztajn, S., Prieto, J., Berasain, C.and Avila, M.A. (2008) The epidermal growth factor receptor ligandamphiregulin participates in the development of mouse liver fibrosis.Hepatology 48 (4), 1251–1261.

[24] Oakley, F., Mann, J., Nailard, S., Smart, D.E., Mungalsingh, N., Constandinou, C.,Ali, S., Wilson, S.J., Millward-Sadler, H., Iredale, J.P. and Mann, D.A. (2005)Nuclear factor-jB1 (p50) limits the inflammatory and fibrogenic responses tochronic injury. Am. J. Pathol. 166, 695–708.

[25] Nelson, W., Tong, Y.L., Lee, J.K. and Halberg, F. (1979) Methods for cosinorrhythmometry. Chronobiologia 6, 305–323.

[26] Martino, T.A., Tata, N., Belsham, D.D., Chalmers, J., Straume, M., Lee, P., Pribiag,H., Khaper, N., Liu, P.P., Dawood, F., Backx, P.H., Ralph, M.R. and Sole, M.J.(2007) Disturbed diurnal rhythm alters gene expression and exacerbatescardiovascular disease with rescue by resynchronization. Hypertension 49,1104–1113.

[27] Chen, P., Li, C., Pang, W., Zhao, Y., Dong, W., Wang, S. and Zhang, J. (2009) Theprotective role of Per2 against carbon tetrachloride-induced hepatotoxicity.Am. J. Pathol. 174 (1), 63–70.

[28] Oishi, K., Shirai, H. and Ishida, N. (2005) CLOCK is involved in the circadiantransactivation of peroxisome-proliferator-activated receptor a (PPARa) inmice. Biochem. J. 386, 575–581.

[29] Oishi, K., Miyazaki, K., Kadota, K., Kikuno, R., Nagase, T., Atsumi, G., Ohkura, N.,Azama, T., Mesaki, M., Yukimasa, S., Kobayashi, H., Iitaka, C., Umehara, T.,Horikoshi, M., Kudo, T., Shimizu, Y., Yano, M., Monden, M., Machida, K.,Matsuda, J., Horie, S., Todo, T. and Ishida, N. (2003) Genome-wide expressionanalysis of mouse liver reveals CLOCK-regulated circadian output genes. J.Biol. Chem. 278 (42), 41519–41527.

[30] Desvergne, B. and Wahli, W. (1999) Peroxisome proliferator-activatedreceptors: nuclear control of metabolism. Endocr. Rev. 20 (5), 649–688.

[31] Wang, X.J., Chamberlain, M., Vassieva, O., Henderson, C.J. and Wolf, C.R. (2005)Relationship between hepatic phenotype and changes in gene expression incytochrome P450 reductase (POR) null mice. Biochem. J. 388, 857–867.

[32] Djouadi, F., Weinheimer, C.J., Saffitz, J.E., Pitchford, C., Bastin, J., Gonzalez, F.J.and Kelly, D.P. (1998) A gender-related defect in lipid metabolism and glucosehomeostasis in peroxisome proliferator-a activated receptor alpha- deficientmice. J. Clin. Invest. 102 (6), 1083–1091.

[33] MacDonald, G.A., Bridle, K.R., Ward, P.J., Walker, N.I., Houglum, K., George,D.K., Smith, J.L., Powell, L.W., Crawford, D.H. and Ramm, G.A. (2001) Lipidperoxidation in hepatic steatosis in humans is associated with hepatic fibrosisand occurs predominately in acinar zone 3. J. Gastroenterol. Hepatol. 16, 599–606.

[34] Barreiro, P., Rodriguez-Novoa, S., Labarga, P., Ruiz, A., Jimenez-Nacher, I.,Martin-Carbonero, L., Gonzalez-Lahoz, J. and Soriano, V. (2007) Influence ofliver fibrosis stage on plasma levels of antiretroviral drugs in HIV-infectedpatients with chronic hepatitis C. J. Infect. Dis. 195, 973–979.