Modulation of circadian expression of D-site binding protein by the schedule of parenteral nutrition...

Modulation of Circadian Expression of D-Site Binding Protein bythe Schedule of Parenteral Nutrition in Rat Liver

ATSUHIRO OGAWA,1 MASAHIKO YANO,1 TOSHIMASA TSUJINAKA,1 TAKASHI MORIMOTO,1 SHUNJI MORITA,1

MASAAKI TANIGUCHI,1 HITOSHI SHIOZAKI,1 KEIICHI OKAMOTO,2 SEIJI SATO,2 AND MORITO MONDEN1

The aim of this study was to investigate the changes in ing and peaks at approximately 8 PM in rat liver.4 The circa-dian rhythm of DBP is reported to be independent of externalthe circadian rhythm of the expression of liver-specific genes

caused by different schedules of parenteral nutrition (PN). stimuli, such as constant lighting or darkness, starvation, orwater deprivation.4 DBP interacts with promoter regions andRats received PN continuously throughout the day or intermit-

tently during the night or day for 7 days. They were examined activates the transcription of several genes, including thealbumin gene3 and the cytochrome P450 cholesterol 7a-hy-for gene expression of D-site binding protein (DBP), albumin,

and cytochrome P450 cholesterol 7a-hydroxylase (CYP7) in droxylase (CYP7) gene,5,6 a rate-limiting enzyme in the con-version of cholesterol to bile acid. CYP7 also follows a circa-the liver. The nocturnal PN group showed circadian expres-

sion of DBP messenger RNA (mRNA) and protein with a peak dian rhythm similar to DBP, suggesting the involvement ofDBP in circadian cholesterol homeostasis.at 10 PM, in the same manner as the control rats receiving

normal chow feeding. However, the diurnal PN group showed In most animals, circadian rhythms can be seen in behav-ior, blood hormone levels, and enzyme activities.1 When theinverted expression of DBP mRNA and protein with a peak

at 10 AM. CYP7 mRNA levels exhibited good synchronization daily feeding schedule is restricted, a wide range of rhythmicbiological functions is known to be modulated in rodents.1with the levels of DBP mRNA in all groups, whereas albumin

mRNA levels did not show such synchronization. Gel mobil- It was therefore hypothesized that the feeding schedule maymodulate the expression of liver-specific proteins and, as aity-shift assay disclosed that the binding activity of the nuclear

extracts to the CYP7 gene promoter was changed by the PN result, modulate hepatic function.In this study, we investigated the effects of different sched-schedule in accordance with the expression of CYP7 mRNA.

The PN schedule modulates the circadian rhythm of DBP ules of parenteral nutrition (PN) on the hepatic biologicalcircadian rhythm by examining the expression of the liver-expression and may have an effect on hepatic bile acid forma-

tion through transcriptional regulation of the CYP7 gene. enriched transcription factor, DBP, and the liver-specificgenes, CYP7 and albumin.(HEPATOLOGY 1997;26:1580-1586.)

MATERIALS AND METHODSA variety of biological functions are known to possess

Animals. Six-week-old male Donryu rats were obtained from SLC,circadian rhythms and to be regulated by the circadian clock.1Hamamatsu, Japan, housed individually, and maintained on a 12-Hepatic functions, such as protein synthesis and enzymatichour light-dark cycle (light: 7 AM-7 PM; dark: 7 PM-7 AM). Underactivities, also exhibit diurnal changes, though the exactpentobarbital anesthesia, a silastic catheter (0.5-mm inner diameter;mechanisms are not known.Dow Corning, Midland, MI) for PN was inserted through the right

Recent advances in molecular biology have revealed that jugular vein with the tip directed toward the right atrium. Thenumerous genes encoding many liver-specific proteins and catheter was fixed to the vein and exteriorized through an incisionenzymes are regulated at the transcriptional level.2 This regu- between the scapulae, according to the method of Steiger et al.7 Thelation is controlled by liver-enriched transcription factors, rats were divided into three groups. The first received a standard PN

solution (250 kcal/kg/d, 1.78 gN/kg/d) administered continuouslyamong which is the D-site binding protein (DBP). DBP wasthroughout the day at a rate of 240 mL/d/kg (continuous PN). Theoriginally described as a factor binding to the albumin D-site3

standard PN solution was composed of GE-3 and Amiparen (Otsukaand is known to maintain a stringent circadian expressionPharmacy Co., Tokushima, Japan), 216.4 g/L glucose, and 47.1 g/rhythm; the level of DBP is almost undetectable in the morn-L amino acid. The two other groups received the same PN regimenat double the rate of the continuous PN group for a restricted time,either during the day from 8 AM to 8 PM (diurnal PN) or at nightfrom 8 PM to 8 AM (nocturnal PN). All groups received half-strengthAbbreviations: DBP, D-site binding protein; CYP7, cytochrome P450 cholesterol 7a-PN for the first 24 hours, followed by full-strength PN for thehydroxylase; PN, parenteral nutrition; cDNA, complementary DNA; C/EBPa, CCAAT

enhancer binding protein a; C/EBPb, CCAAT enhancer binding protein b; mRNA, following 6 days. They were not given anything orally for the dura-messenger RNA; TEN, total enteral nutrition. tion of the experiment. On the seventh day, the rats were killed at

From the 1Department of Surgery II, Osaka University Medical School, Suita, Osaka, specific times (4 AM, 10 AM, 4 PM, 10 PM) under pentobarbitaland 2Pharmacology Section, Nutrition Research Institute, Otsuka Pharmaceutical Fac- anesthesia, and the livers were removed. As the control, the liverstory, Inc., Naruto, Tokushima, Japan. were obtained from rats given a standard commercial nonpurified

Received May 23, 1997; accepted August 13, 1997.diet (Oriental East Co., Ltd., Tokyo, Japan) and water ad libitum.

Address reprint requests to: Masahiko Yano, M.D., Department of Surgery II, OsakaBlood samples were collected from the abdominal aorta to measureUniversity Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Fax: 81-6-879-serum albumin and corticosterone. All procedures were approved3259.by the Committee for Use of Laboratory Animals at Osaka Univer-Copyright q 1997 by the American Association for the Study of Liver Diseases.

0270-9139/97/2606-0029$3.00/0 sity Medical School.

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AID Hepa 0058 / 5p2a$$1141 11-21-97 10:19:18 hepa WBS: Hepatology

HEPATOLOGY Vol. 26, No. 6, 1997 OGAWA ET AL. 1581

FIG. 1. Effect of PN schedule on DBP mRNA levels in rat liver. Northern blot analysis was performed on total RNA (10 mg) extracted at 4 AM, 10AM, 4 PM, and 10 PM from livers of normally fed rats (FED) (n Å 4) and those receiving continuous PN (n Å 4), nocturnal PN (n Å 4), and diurnal PN(n Å 4) for 7 days. Autoradiograms were densitometrically analyzed and normalized with b-actin mRNA levels. The results are expressed as percentagesof the control and as the mean { SEM of four rats in each group. Normally fed rats killed at 10 PM were used as controls (100%). If the SEM bar is notvisible, it is smaller than the symbol size. The respective bands of the autoradiogram are shown above the graph. Diurnal variations were examined byANOVA, and a difference of P õ .05 was considered significant.

Preparation of Total RNA. Total RNA from the frozen liver samples Noshiro, Department of Biochemistry, Hiroshima University Schoolof Dentistry, Hiroshima, Japan. The rat albumin cDNA probe waswas isolated by the method of Chomczynski and Sacchi8 using

ISOGEN (Nippon Gene, Tokyo, Japan). The concentration of RNA a kind gift from K. Sugiyama, National Cancer Center InstituteEast, Chiba, Japan. The probes, digested from cloned cDNAs, werewas determined by measuring the absorbance at 260 nm.

Northern Blot Analysis. Northern blot was performed essentially radiolabeled by the random primer synthesis method using a com-mercial kit (Megaprime DNA labeling system, Amersham Interna-as described previously.9 Total RNA (10 mg/lane) was separated by

electrophoresis on 1% agarose gel and then transferred to a nylon tional) according to the manufacturer’s instructions. The nylonmembrane filters were hybridized with the respective probes by themembrane (Hybond N/, Amersham International, Buckingham-

shire, England). Complementary DNA (cDNA) probes of rat DBP, method of Church.12 The filters were then washed twice in 21saline sodium citrate for 15 minutes, in 11 saline sodium citraterat CCAAT/enhancer binding protein a (C/EBPa),10 rat CCAAT/

enhancer binding protein b (C/EBPb)11 and mouse b-actin were for 60 minutes, and in 0.51 saline sodium citrate for 30 minutesat 657C. After the washing, the filters were exposed to autoradiogra-generously provided by F. J. Gonzalez, Laboratory of Molecular

Carcinogenesis, National Cancer Institute, Bethesda, MD. The rat phy on Kodak XAR-5 film (Eastman Kodak, Rochester, NY). Theabundance of messenger RNAs (mRNAs) was determined by densi-cholesterol 7a-hydroxylase cDNA probe was kindly donated by M.

FIG. 2. Effect of the PN schedule on C/EBPa mRNA levels in rat liver. Northern blot analysis was performed on total RNA (10 mg) extracted at 4AM, 10 AM, 4 PM, and 10 PM from livers of normally fed rats (FED) (n Å 4) and those receiving continuous PN (n Å 4), nocturnal PN (n Å 4), anddiurnal PN (n Å 4) for 7 days. Autoradiograms were densitometrically analyzed and normalized with b-actin mRNA levels. The results are expressed aspercentages of the control and as the mean { SEM of four rats in each group. Normally fed rats killed at 10 PM were used as controls (100%). If theSEM bar is not visible, it is smaller than the symbol size. The respective bands of the autoradiogram are shown above the graph. Diurnal variations wereexamined by ANOVA, and a difference of P õ .05 was considered significant.


1582 OGAWA ET AL. HEPATOLOGY December 1997

FIG. 3. Effect of the PN schedule on C/EBPb mRNA levels in rat liver. Northern blot analysis was performed on total RNA (10 mg) extracted at 4AM, 10 AM, 4 PM, and 10 PM from livers of normally fed rats (FED) (n Å 4) and those receiving continuous PN (n Å 4), nocturnal PN (n Å 4), anddiurnal PN (n Å 4) for 7 days. Autoradiograms were densitometrically analyzed and normalized with b-actin mRNA levels. The results are expressed aspercentages of the control and as the mean { SEM of four rats in each group. Normally fed rats killed at 10 PM were used as controls (100%). If theSEM bar is not visible, it is smaller than the symbol size. The respective bands of the autoradiogram are shown above the graph. Diurnal variations wereexamined by ANOVA, and a difference of P õ .05 was considered significant.

tometric analysis of the film with the MCID system (Imaging Re- and stained with 125I-labeled protein A (Amersham Japan, Tokyo,Japan).search Inc., St. Catherines, Ontario, Canada), and each mRNA abun-

dance was normalized for the b-actin mRNA abundance in the Measurement of Serum Corticosterone Levels. The serum corticoste-rone levels were determined with by method of West et al.18 usingblots. The linearity of the densitometric analysis had been confirmed

previously.9 Data for each group were analyzed by ANOVA to deter- radioimmunoassay. Data for each group were analyzed by ANOVAto determine whether there were any significant diurnal variations.mine whether there were any significant diurnal variations. Differ-

ences of P õ .05 were considered significant. Differences of P õ .05 were considered significant.Preparation of Nuclear Extracts. Rat liver nuclear extracts were pre-

RESULTSpared according to the method of Gorski et al.,13 essentially asdescribed previously.14 Extracts were prepared from rats killed at We first examined the effects of the PN schedule on the10 AM or 10 PM. mRNA levels of the liver-enriched nuclear transcription fac-

Western Blot Analysis. Nuclear extract (20 mg) separated by so- tors, DBP, C/EBPa, and C/EBPb. C/EBPa and C/EBPb aredium dodecyl sulfate–polyacrylamide gel electrophoresis in a 10- members of the bZIP family of transcription factors and regu-20 gradient gel (Daiichi Pure Chemicals Co., Tokyo, Japan) was late the expression of various genes in the liver.2 In controltransferred electrophoretically to an Immobilon-P membrane (Milli-

rats receiving normal feeding and water ad libitum, DBPpore Co., Madison, WI) with a semidry Trans-Blot (Bio-Rad Labora-mRNA levels showed a stringent circadian rhythm, as hastories, Hercules, CA). After the blocking, the membrane was treatedbeen found in another study.4 It was undetectable in thewith rabbit anti-rat C/EBPa, anti-rat C/EBPb (both from Santa Cruzdaytime and reached a peak at 10 PM (Fig. 1). The continuousBiotechnology, Inc., Santa Cruz, CA) or anti-rat DBP antibody, pro-

vided from Dr. U. Schibler, Department of Molecular Biology, Uni- PN group showed a similar circadian pattern of DBP mRNAversity of Geneva, Geneva, Switzerland, and stained by the alkaline expression, though the circadian change was very minute.phosphatase reaction using a Proto-Blot system (Promega Co., Mad- In the nocturnal PN group, the rhythm of DBP mRNA expres-ison, WI). sion was as stringent as that in the control rats, while the

Gel Mobility-Shift Analysis. Gel mobility-shift assays were per- diurnal PN group showed an inverted circadian rhythm withformed by the method of Cereghini et al.,15 essentially as described a peak at 10 AM. C/EBPa (Fig. 2) and C/EBPb (Fig. 3) mRNApreviously.14 The double-stranded synthetic oligonucleotides used

showed no significant rhythms in the control rats or any offor the CYP7-FP2 site5 were: 5*-CTTTGGATGTTATGTCAGCAC-the three PN groups.ATGACTTTC-3*, and those for the albumin-D site16 were: 5*-TGG-

Western blot analysis using the nuclear extracts preparedTATGATTTTGTAATGGGG-3*.at 10 AM and 10 PM showed a circadian rhythm of DBP32P-labeled oligonucleotides at 0.05 ng (20 ng of unlabeled oligo-

nucleotides for gel mobility-shift Western immunoblot analysis) protein levels. DBP protein was identified clearly at 10 PMwere incubated with 20 mg of nuclear extract proteins for 30 min- but was undetectable at 10 AM in the nocturnal PN groups,utes at 247C in 20 mL of the binding buffer as described by Ueno and an inverted pattern was observed in the diurnal PN groupand Gonzalez.17 After incubation, the mixtures were subjected to (Fig. 4). C/EBPa and C/EBPb proteins did not show anyelectrophoresis in a 5% nondenaturing acrylamide gel. The gels diurnal variations in any of the three PN groups.were then soaked for 20 minutes in 5% glycerol, dried, and exposed Because DBP has been known to transactivate both theto Kodak XAR-5 X-ray film at 0707C.

CYP75 and the albumin3 gene promoters in vitro, we exam-The specificity of the binding was confirmed by the addition ofined the effects of the PN schedule on the mRNA levels ofunlabeled oligonucleotides as a competitor.these two genes. CYP7 mRNA levels showed the sameThe gel containing unlabeled oligonucleotides was transferred tochanges as the levels of DBP mRNA (Fig. 5). In control rats,a nitrocellulose membrane (Life Technologies, Inc., Gaithersburg,

MD). The filters were treated with rabbit anti-rat DBP antibody the CYP7 mRNA level showed a circadian rhythm as has



tive genes. Gel mobility-shift analysis showed the bindingactivity to the CYP7-FP2 site was high at 10 PM and low at10 AM in the nocturnal PN group. In contrast, no such changewas found in the diurnal PN group (Fig. 7A). The bindingactivity to the albumin D-site did not show any significantcircadian changes in both PN groups (Fig. 7B).

For the CYP7-FP2 site, DBP and C/EBPb protein can bindto modulate and regulate the transcription of the CYP7gene.5,6 The retarded bands of FP2-site oligomer may thusconsist of DBP and C/EBPb. Therefore, to examine theinvolvement of DBP in the retarded FP2-site oligomer, gelmobility-shift Western blot analysis was performed using theantibody for DBP protein (Fig. 8). Gel mobility-shift Westernblot analysis showed the binding activity of DBP to the CYP7-FP2 site was high at 10 PM and low at 10 AM in the nocturnalPN group. In contrast, no such change was found in theFIG. 4. Effect of the PN schedule on protein levels of DBP, C/EBPa, and

C/EBPb in rat liver nuclear extracts. Western blot analysis was performed using diurnal PN group.the liver nuclear extracts (20 mg) obtained either at 10 AM or 10 PM from rats These results showed that the PN schedule modulated thereceiving continuous PN, nocturnal PN and diurnal PN for 7 days. circadian expression of the liver-specific nuclear transcrip-

tion factor, DBP, and its target gene, CYP7. However, themechanism remains unknown regarding the regulation ofbeen reported.19 It showed a peak at 10 PM and a trough atthe circadian expression of DBP in the liver. Glucocorticoid10 AM. In the continuous PN group, no circadian rhythm ofhormone is one of the candidates for a regulator of DBPCYP7 mRNA expression could be identified. In the nocturnaltranscription, because blood glucocorticoid levels show a cir-PN group, the circadian change was as evident as that ob-cadian oscillation20 and the promoter of the DBP gene con-served in the control rats, while the diurnal PN group showedtains a glucocorticoid-responsive element.21 Therefore, wean inverted circadian rhythm of CYP7 mRNA expression withexamined serum corticosterone levels in the three PN groupsa peak at 10 AM.(Fig. 9). A circadian oscillation was found only in the noctur-In contrast to the CYP7 mRNA levels, the albumin mRNAnal PN groups, and serum corticosterone levels did not corre-levels did not show any significant rhythmic changes in thespond to DBP and CYP7 mRNA levels in these three PNcontrol rats or any of the three PN groups (Fig. 6). Thisgroups.agrees with the report of Wuarin and Schibler that the level

of albumin mRNA showed no significant rhythm because ofDISCUSSION

its long half-life.4

In the next step, we examined the binding activity of nu- Rodents are thought to possess a clock determining circa-dian rhythm in the suprachiasmatic nuclei in the hypothala-clear extracts prepared from rats receiving different PN

schedules to the CYP7-FP2 site5 and albumin D-site,3 both mus.22 Circadian rhythms in animals can be found in behav-ior represented by sleeping, food intake, and hormone levels,of which are known as the DBP binding sites of their respec-

FIG. 5. Effect of the PN schedule on CYP7 mRNA levels in rat liver. Northern blot analysis was performed on total RNA (10 mg) extracted at 4 AM,10 AM, 4 PM, and 10 PM from livers of normally fed rats (FED) (n Å 4) and those receiving continuous PN (n Å 4), nocturnal PN (n Å 4), and diurnalPN (nÅ 4) for 7 days. Autoradiograms were densitometrically analyzed and normalized with b-actin mRNA levels. The results are expressed as percentagesof the control and as the mean { SEM of four rats in each group. Normally fed rats killed at 10 PM were used as controls (100%). If the SEM bar is notvisible, it is smaller than the symbol size. The respective bands of the autoradiogram are shown above the graph. Diurnal variations were examined byANOVA, and a difference of P õ .05 was considered significant.



FIG. 6. Effect of the PN schedule on albumin mRNA levels in rat liver. Northern blot analysis was performed on total RNA (10 mg) extracted at 4AM, 10 AM, 4 PM, and 10 PM from livers of normally fed rats (FED) (n Å 4) and those receiving continuous PN (n Å 4), nocturnal PN (n Å 4), anddiurnal PN (n Å 4) for 7 days. Autoradiograms were densitometrically analyzed and normalized with b-actin mRNA levels. The results are expressed aspercentages of the control and as the mean { SEM of four rats in each group. Normally fed rats killed at 10 PM were used as controls (100%). If theSEM bar is not visible, it is smaller than the symbol size. The respective bands of the autoradiogram are shown above the graph. Diurnal variations wereexamined by ANOVA, and a difference of P õ .05 was considered significant.

typically seen in serum levels of glucocorticoid. Destruction consequently control the transcription of a set of genes trans-activated by DBP. We hypothesized that, if destruction of theof the suprachiasmatic nuclei is associated with a loss of the

circadian rhythm of the adrenal corticosterone level,23 as well suprachiasmatic nuclei causes a change in food intake pat-tern, the nutritional schedule would play a role in the circa-as modification of the DBP mRNA rhythm.24 Glucocorticoid

may regulate the circadian rhythm of DBP expression and dian change of the glucocorticoid level or DBP expression.

FIG. 7. Effect of the PN sched-ule on binding activity of the CYP7-FP2 site (A) or albumin D-site (B)in rat liver nuclear extracts. Gel mo-bility-shift analysis was performedusing liver nuclear extracts (20 mg)obtained either at 10 AM or 10 PM

from rats receiving nocturnal PN ordiurnal PN for 7 days, respectively.Arrows indicate the bands of nuclearproteins that bind to the oligonucle-otides. Competition analysis wasperformed by adding nonlabeled oli-gonucleotide to confirm the speci-ficity of the bands (com).



also rats receiving PN with different schedules, although nocorrelation was found with the mRNA and protein levels ofC/EBPb. These results suggested that the circadian expres-sion of CYP7 mRNA is mainly regulated by DBP in vivo.Bile acid formation may undergo a circadian change, andnocturnal PN may restore the normal pattern of bile acidformation seen with oral feeding, because the diurnal changein CYP7 mRNA was the same as that observed with oralfeeding.

In contrast to CYP7 mRNA, the albumin mRNA, the tran-scription of which is also partially controlled by DBP,3 didnot show any rhythm in any of the nutrition schedules. Thereason for this may be that its half-life is long enough to notexhibit a circadian rhythm, or the binding of DBP to thealbumin gene is not as specific as to the CYP7 gene. Gelmobility-shift analysis was thus conducted to elucidate thebinding activity of the nuclear extracts to the DBP bindingsites of the CYP7 and albumin genes. Signal intensities ofretarded bands in the CYP7-FP2 site correlated with the DBPprotein levels at both the nocturnal and diurnal PN groups.However, no such correlation was seen at the albumin D-site, which may have a binding specificity weaker for DBPthan for CYP7-FP2, because not only DBP, but also C/EBPa10

and C/EBPb11 can bind to it. These findings showed that DBPdoes not induce a circadian change in albumin transcription.Furthermore, the binding activity of DBP to the CYP7-FP2site was confirmed by the gel mobility-shift Western blotanalysis using anti-DBP antibody.

We thus showed that the schedule of PN can modify thecircadian rhythm of DBP. The next question was how theschedule of nutrition has an effect on DBP expression. Severallines of evidence suggested the role of glucocorticoid in DBPexpression.21 Its circadian nature may be controlled by thecyclic secretion of glucocorticoid. In hepatoma cells, the ad-

FIG. 8. Effect of the PN schedule on binding activity of DBP to thedition of dexamethasone leads to rapid extinction of DBPCYP7-FP2 site in rat liver nuclear extracts. Gel mobility-shift Western blotmRNA accumulation.21 Injection of dexamethasone to ratsanalysis was performed using liver nuclear extracts (20 mg) obtained either

at 10 AM or 10 PM from rats receiving nocturnal PN or diurnal PN for 7 induces strong attenuation of DBP mRNA accumulation,4days, respectively. The bands showed DBP proteins that bind to the CYP7- and the promoter region of the DBP gene contains a glucocor-FP2 site. ticoid-responsive element.21 Blood glucocorticoid levels

show a circadian rhythm with a peak in the evening justbefore the light/dark switch in normally fed rats.20

We hypothesized that the schedule of PN may modulatePreviously, we observed that the mRNA level of albumin inthe liver was low in rats receiving continuous PN compared serum glucocorticoid levels, through which the expression

of DBP may be controlled. The results of the present studywith rats receiving oral feeding, and that the level was re-stored by the nocturnal cyclic PN.25 Because the albumin showed that serum corticosterone levels showed a clear circa-gene is known to be transactivated by DBP,3 a schedule ofartificial nutrition may regulate the circadian rhythm of DBPand affect transcription of the target gene.

The results of the present study revealed for the first timethat the circadian rhythm of DBP expression is modulatedby the schedule of nutrition. Nocturnal PN resembles thenormal feeding habit because rats eat at night and sleep dur-ing the day. This group maintained the circadian rhythm ofthe DBP mRNA level with a peak at 10 PM as observed withoral feeding. However, in the diurnal PN group, the rhythmof DBP mRNA expression was completely inverted with apeak at 10 AM. Western blot analysis showed that the changesin DBP mRNA levels corresponded with those in protein

FIG. 9. Effect of the PN schedule on serum corticosterone levels in rats.levels.Blood samples were obtained at 4 AM, 10 AM, 4 PM, and 10 PM from ratsThe 5* proximal promoter region of the CYP7 gene con-receiving continuous PN (n Å 4), nocturnal PN (n Å 4), and diurnal PNtains several binding sites for DBP and C/EBPb.5,6 The diur- (n Å 4) for 7 days. Results are expressed as mean { SEM of four rats in

nal CYP7 mRNA expression correlated well with the mRNA each group. Diurnal variations were examined by ANOVA, and a differenceof P õ .05 was considered significant.and protein levels of DBP in not only normally fed rats, but



M, et al. Regulation of albumin synthesis after hepatectomy and in thedian rhythm in the nocturnal PN group as seen in rats receiv-acute inflammation phase of rat liver. J Nutr Biochem 1995;6:522-527.ing normal feeding. Loss of the circadian rhythm was ob-

10. Friedman AD, Landschulz WH, Mcknight SL. C/EBP activates the pro-served in the continuous and diurnal PN groups. Saito et al. moter of the serum albumin gene in cultured hepatoma cells. Genesshowed that a total enteral nutrition (TEN) schedule for 4 Dev 1989;3:1314-1322.

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from the mouse albumin promoter. Cell 1986;47:767-776.shows an inverted circadian rhythm. The PN schedule in this14. Yano M, Falvey E, Gonzalez FJ. Role of the liver-enriched transcriptionstudy might not have been long enough to allow synchroniza-

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