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The Journal of Nutrition Vol. 127 No. 7 July 1997, pp. 1328-1332
Copyright ©1997 by the American Society for Nutritional Sciences

Gene Expression of Albumin and Liver-Specific Nuclear Transcription Factors in Liver of Protein-Deprived Rats1,2

Atsuhiro Ogawa3, Masahiko Yano, Toshimasa Tsujinaka, Chikara Ebisui,, Takashi Morimoto, Masanori Kishibuchi, Junya Fujita, Syunji Morita, Hitoshi Shiozaki, and Morito Monden

Department of Surgery II, Osaka University Medical School, Suita, Osaka, 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED

ABSTRACT

Protein-energy malnutrition causes hypoalbuminemia. Recent work has suggested that this may be partly due to decreased transcription of the albumin gene. This study examined the role of cis-acting and trans-acting elements of the albumin gene during protein deprivation. Male 7-wk-old Donryu rats were fed a protein-free diet (0% casein diet) for 10 d or given restricted (pair-fed control) or free access (freely fed control) to a 25% casein diet. Serum albumin concentrations were significantly lower in the protein-deprived rats (29 ± 1 g/L) than in the pair-fed controls (42 ± 3 g/L) or the freely fed controls (45 ± 3 g/L). The albumin mRNA level was also significantly lower in livers of protein-deprived rats (36% of pair-fed control). However, gel mobility shift analysis using liver nuclear extracts did not show any significant difference between the protein-deprived rats and the pair-fed controls in the binding activity to the B and D sites of the albumin promoter. Furthermore, gel mobility shift-Western blot analysis showed no significant difference between the two groups in the protein levels of nuclear transcription factors binding to the D sites. The amounts of mRNA of hepatocyte nuclear factor-1 binding to the B site were not significantly different between these two groups. These results suggest that the proximal promoter region may not play a major role in the down-regulation of the albumin gene during protein deprivation.

KEY WORDS: albumin · nuclear transcription factors · protein deprivation · rats

INTRODUCTION

Albumin is a major secretory protein of the liver, and its synthesis varies under different clinical conditions (Doweiko and Nompleggi 1991a and 1991b). Nutritional status has a profound effect on the overall rate of protein synthesis. Hypoalbuminemia is often seen in protein-energy malnutrition, but not when energy alone is restricted (Enwonwu and Sreebny 1971). In the former condition, not only the serum albumin level but also the albumin mRNA level is markedly decreased (Sakuma et al. 1987). By using ribonuclease protection assay, Straus et al. (1994) found that reduction in albumin mRNA levels in protein-restricted rats is caused at least partly by a decrease in gene transcription. Recently, an in vitro study with rat hepatoma cells showed that amino acid limitation suppresses albumin gene transcription (Marten et al. 1994). These findings strongly suggested that transcription factors may play an important role in hypoalbuminemia in protein deficiency. Therefore, we examined what transcription factor or factors modulate the expression of the albumin gene during protein deprivation.

The structure and mechanism of the transcriptional regulation of the albumin gene have been clarified recently. The promoter region of the albumin gene consists of six cis-elements, the A to F sites, recognized by corresponding nuclear factors (Lichtsteiner et al. 1987). The factors binding to the B and D sites have been reported to play a key role in the liver-specific expression of the albumin gene (Maire et al. 1989). Hepatocyte nuclear factor-1 (HNF-1)4 binds to the B site (Lichtsteiner and Schibler 1989), and the D site binding protein (DBP) (Mueller et al. 1990), CCAAT/enhancer binding protein (C/EBP) alpha  (Friedman et al. 1989) and C/EBPbeta (Descombes et al. 1990) bind to the D site.

We previously reported that a change in the expression of nuclear transcription factor genes contributes to the down-regulation of albumin gene transcription in the regenerating liver and during inflammation (Morimoto et al. 1995). However, the role of nuclear transcription factors in the expression of albumin genes during protein deficiency is unknown.

This study was designed to examine the involvement of these nuclear transcription factors in the down-regulation of the albumin gene during protein deficiency.

MATERIALS AND METHODS

Animals and diets. Seven-week-old male Donryu rats were obtained from SLC (Hamamatsu, Japan), housed individually, and maintained on a 12-h light:dark cycle. After acclimation for 1 wk, the rats were given isocaloric experimental diets containing 0% (protein-deprived group) or 25% (pair-fed control group) casein (Oriental Yeast Co., Tokyo, Japan) for 10 d (Table 1). Food intake of the protein-deprived group was measured every day, and 25% casein diet in an amount equal to the mean amount of this group was provided on the next day to the pair-fed control group. A third group was given free access to the 25% casein diet (freely fed control group). All rats had free access to water. At the end of the experiment, the rats were weighed and killed by overdose of ether anesthesia. Blood samples were collected from the inferior vena cava to measure serum albumin by the bromocresol green method (Doumas et al. 1971). After liver perfusion with PBS from the portal vein, liver samples were taken. Fresh liver samples were used for extraction of nuclear proteins, and frozen liver samples were stored at -70°C until RNA extraction. All samples were obtained at 2200 h because of the known circadian expression of the transcription factor DBP (Wuarin and Schibler 1990). All studies were approved by the Committee for Use of Laboratory Animals at Osaka University Medical School.

Table 1. Composition of the diets

[View Table]

Preparation of total RNA. Total RNA from the frozen liver samples of the protein-deprived group (n = 7), the pair-fed control group (n = 6) and the freely fed control group (n = 6) was isolated by the method of Chomczynski and Sacchi (1987) using ISOGEN (Nippon Gene, Tokyo, Japan). The RNA concentration was determined by measuring absorbance at 260 nm.

Northern blot analysis. Northern blot analysis was performed essentially as described previously (Morimoto et al. 1995). RNA (20 µg/lane) was separated by electrophoresis on 1% agarose gel and then transferred to a nylon membrane (Hybond N+, Amersham International, Buckinghamshire, UK). The rat albumin cDNA probe was kindly provided by K. Sugiyama, National Cancer Center Institute East, Chiba, Japan. cDNA probes of rat HNF-1 and mouse beta -actin were generously provided by F. J. Gonzalez, Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD. Rat retinol-binding protein (RBP) cDNA probe was generously donated by S. Katoh, Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University of Agriculture, Tokyo, Japan. The probes, digested from cloned cDNAs, were radiolabeled by the random primer synthesis method using a commercial kit (Megaprime DNA labeling system, Amersham International) according to the manufacturer's instructions. The nylon membrane filters were hybridized with the respective probes using the method of Church and Gilbert (1984). The filters were then washed twice in 2× saline-sodium citrate (SSC) for 15 min, in 1× SSC for 60 min, and then in 0.5× SSC for 30 min at 65°C. After the washing, the filters were exposed to autoradiography on Kodak XAR-5 film (Eastman Kodak, Rochester, NY). The amount of mRNA was determined by densitometric analysis of the film with the MCID system (Imaging Research Inc., St. Catherines, Ontario, Canada), and each amount was normalized to the beta -actin mRNA abundance in the blots. The linearity of the densitometric analysis had been confirmed previously (Morimoto et al. 1995).

Preparation of nuclear extracts. Rat liver nuclear extracts of both the protein-deprived group (n = 3) and the pair-fed control group (n = 3) were prepared according to the method of Gorski et al. (1986), essentially as described by Yano et al. (1992). Extract was prepared from rats killed at 2200 h because of the circadian expression of the transcription factor DBP (Wuarin and Schibler 1990).

Gel mobility shift analysis. Gel mobility shift assays were performed by the method of Cereghini et al. (1988) essentially as described by Yano et al. (1992). The following double-stranded synthetic oligonucleotides were used: B site, 5'-GTGGTTAATGATCTACAGTTA: D site, 5'-TGGTATGATTTTGTAATGGGG (Cereghini et al. 1987).

32P-labeled oligonucleotides (0.05 ng, 20 ng of unlabeled oligonucleotides for gel mobility shift-Western immunoblot analysis) were incubated with 10 µg of nuclear extract proteins for 30 min at 24°C in 20 µL binding buffer as described by Ueno and Gonzalez (1990). After incubation, the mixtures were subjected to electrophoresis in a 5% nondenaturing acrylamide gel. The gels were soaked for 20 min in 5% glycerol, dried, and exposed to Kodak XAR-5 X-ray film at -70°C. The gel containing unlabeled oligonucleotides was transferred to a nitrocellulose membrane (Life Technologies, Gaitherburg, MD). The filters were treated with rabbit anti-rat C/EBPalpha , anti-rat C/EBPbeta (both Santa Cruz Biotechnology, Santa Cruz, CA) or anti-rat DBP antibody and stained with 125I-labeled protein A (Amersham Japan, Tokyo, Japan). Rabbit anti-rat DBP antibody was kindly provided by U. Schibler, Department of Molecular Biology, University of Geneva, Geneva, Switzerland. Nuclear protein levels were determined by densitometric analysis of the films as the amount of mRNAs.

Western blot analysis. Nuclear extract (10 µg) separated by SDS-PAGE in a 10-20 gradient gel (Daiichi Pure Chemicals, Tokyo, Japan) was transferred electrophoretically to an Immobilon-P membrane (Millipore, Madison, WI) with a semidry Trans-Blot (Bio-Rad Laboratories, Hercules, CA). After the blocking, the membrane was treated with anti-C/EBPbeta antibodies and anti-rabbit immunoglobulin G conjugated with alkaline phosphatase using a ProtoBlot system (Promega Co., Madison, WI) and stained by the alkaline phosphatase reaction. The levels of nuclear proteins were determined by densitometric analysis of the filters as the abundance of mRNAs.

Statistical analysis. Body weight, mRNA level and serum albumin data were statistically evaluated using ANOVA followed by Scheffé's test (Scheffé 1959) using a computer program (Stat View-J, version 4.11, Abacus Concepts, Berkeley, CA). Nuclear protein data were statistically evaluated using the unpaired t test. Differences of P < 0.05 were considered significant.

RESULTS

Food intake by the protein-deprived group and the pair-fed control group (mean, 16.3 g/d) was approximately 80% of that of the freely fed control group (mean, 20.1 g/d). Body weight was significantly lower in the protein-deprived rats than in the pair-fed controls (Fig. 1). Rats fed the protein-free diet had hypoalbuminemia, but there was no significant difference in the serum albumin concentration between the two control groups (Table 2). The albumin mRNA level in the protein-deprived group was significant lower than that in the pair-fed and freely fed control groups. The control groups did not differ significantly. Our results supported previous reports that both levels of serum albumin and albumin mRNA decrease with protein deprivation but not with energy restriction (Sakuma et al. 1987).
Fig. 1. Body weights in protein-deprived, pair-fed control and freely fed control rats. Seven-week-old Donryu rats were fed the control or the protein-free diet for 10 d. Values are means ± SEM, n = 6 (n = 7 for protein-deprived group). Values at a time point with different letters are significantly different (P < 0.05, Scheffé's test).
[View Larger Version of this Image (22K GIF file)]

Table 2. Serum albumin and hepatic mRNA abundance in freely fed control, pair-fed control and protein-deprived rats1

[View Table]

Interestingly, however, the hepatic mRNA level of the retinol-binding protein (RBP), another nutritional index protein, did not differ among the three groups (Table 2), suggesting that protein deprivation may have some specific effect on hepatic albumin mRNA levels.

To examine the role of nuclear transcription factors in the down-regulation of albumin gene expression during protein deprivation, we performed gel-shift analysis to investigate the binding activity of nuclear proteins for the albumin promoter region, i.e., the B and D sites (Fig. 2). The experiment using nuclear extracts was performed on the pair-fed control group and the protein-deprived group, because no significant difference had been found in the levels of serum albumin and albumin mRNA between the two control groups. Gel-shift analysis revealed no major difference in the amount of retarded bands of B site oligomer and D site oligomer between the pair-fed control and protein-deprived groups.

Fig. 2. Gel mobility shift analysis of liver nuclear extract isolated from pair-fed control or protein-deprived rats. Seven-week-old Donryu rats were fed the control diet (CON, lanes 2-4) or the protein-free diet (PF, lanes 5-7) for 10 d. At the end of the experiment, liver nuclear extracts were obtained. 32P-labeled oligonucleotides (0.20 ng) for the B site (panel A) or the D site (panel B) of the albumin promoter were incubated with 10 µg of liver nuclear extract and subjected to 5% acrylamide gel electrophoresis and autoradiography. The slow migration band is a construct of oligonucleotides to which nuclear proteins are bound (B), and the fast migration band is a construct of free oligonucleotides (F ). Binding specificity was confirmed because incubation with 200 ng of a nonlabeled oligonucleotide as a competitor caused the band of the bound oligonucleotide to disappear (lane 1). Panel C: Densitometric scans of the results shown in panels A and B, expressed as the relative amount (%) of that of the pair-fed control group. Values are means ± SEM, n = 3.
[View Larger Version of this Image (40K GIF file)]

For the D site, C/EBPalpha , C/EBPbeta and DBP protein can bind in homo- or heterodimers to modulate and regulate the transcription of the albumin gene. The retarded bands of oligomer D may thus consist of C/EBPalpha , C/EBPbeta and DBP. Therefore, to examine the involvement of each nuclear protein that binds to the D site in the retarded D site oligomer, we performed gel shift-Western blot analysis using the antibody for each nuclear protein (Fig. 3). However, this analysis did not show any significant differences in the protein abundance of C/EBPalpha , C/EBPbeta or DBP binding to the D site in the two groups.

Fig. 3. Gel mobility shift-Western blot analysis of CCAAT/enhancer binding protein alpha  (C/EBPalpha ), C/EBPbeta and D site binding protein (DBP) in liver nuclear extract isolated from pair-fed control or protein-deprived rats. Panel A: Seven-week-old Donryu rats were fed the control diet (CON, lanes 1-3) or the protein-free diet (PF, lanes 4-6) for 10 d. At the end of the experiment, liver nuclear extracts were obtained. Unlabeled oligonucleotide (20 ng) for the D site of the albumin promoter was incubated with 10 µg of liver nuclear extract and subjected to 5% acrylamide gel electrophoresis. The gel was then electroblotted on nitrocellulose paper and treated with anti-C/EBPalpha , anti-C/EBPbeta or DBP antibody, then stained by 125I-labeled protein A. Panel B: Densitometric scans of the results shown in panel A, expressed as relative amount (%) of that of the control group. Values are means ± SEM, n = 3.
[View Larger Version of this Image (30K GIF file)]

The same C/EBPbeta mRNA is translated into two different proteins, liver-enriched transcriptional activator protein (LAP) and liver-enriched transcriptional inhibitory protein (LIP) (Descombes and Schibler 1991). Because LIP lacks transcriptional activation domain, it acts as a transcriptional inhibitor. Therefore, a decrease in the LAP/LIP ratio could decrease the transcriptional activity of the albumin gene. We performed Western blot analysis to distinguish LAP from LIP, because they could not be distinguished by gel shift-Western blot analysis (Fig. 4). However, we could not detect LIP, perhaps because the C/EBPbeta antibody we used did not cross-react with LIP or because its amount was very small. The faint band that appeared at 30 kDa is not LIP, because the molecular weight of LIP is 20 kDa.

Fig. 4. Western blot analysis of CCAAT/enhancer binding protein beta  (C/EBPbeta ) in liver nuclear extract isolated from pair-fed control or protein-deprived rats. Panel A: Seven-week-old Donryu rats were fed the control diet (CON, lanes 1-3) or the protein-free diet (PF, lanes 4-6) for 10 d. At the end of the experiment, liver nuclear extracts were obtained. The extracts (10 µg) were separated by SDS-PAGE using a 10-20 gradient gel. The gels were then electroblotted onto an Immobilon-P membrane and treated with anti-C/EBPbeta antibody and anti-rabbit immunoglobulin G conjugated with alkaline phosphatase and stained using the alkaline phosphatase reaction. Panel B: Densitometric scans of the results shown in panel A, expressed as relative amount (%) of that of the control group. Values are means ± SEM, n = 3.
[View Larger Version of this Image (27K GIF file)]

Hepatocyte nuclear factor-1 is the only protein reported to bind to the B site (Lichtsteiner and Schibler 1989). Instead of measuring the amount of HNF-1 protein, we performed Northern blot analysis to estimate the amount of the HNF-1 mRNA, because we could not obtain the anti-HNF-1 antibody. There was no significant difference between the pair-fed control group and the protein-deprived group (Fig. 5).

Fig. 5. Northern blot analysis of hepatocyte nuclear factor-1 (HNF-1) in liver isolated from pair-fed control or protein-deprived rats. Panel A: Seven-week-old Donryu rats were fed the control diet (CON, lanes 1-6) or the protein-free diet (PF, lanes 7-13) for 10 d. Panel B: Densitometric scans of the results of Northern blot analysis were normalized for beta -actin mRNA amount. All data are expressed as relative amount (%) of that of the pair-fed control group. Values are means ± SEM, n = 6 for the pair-fed control group, n = 7 for the protein-deprived group.
[View Larger Version of this Image (35K GIF file)]

DISCUSSION

The serum albumin concentration is the net result of synthesis, distribution and degradation. Many factors influence albumin synthesis, such as hormones, stress, nutrition, colloid osmotic pressure, and presense of tumor (Doweiko and Nompleggi 1991a and 1991b). Recent advances in molecular biology have revealed that changes in albumin synthesis under these pathophysiological conditions may in part be due to transcriptional regulation of the albumin gene.

Previously, we reported that the albumin mRNA level is down-regulated in hepatectomized rats as well as in a model of rat inflammation induced by turpentine oil injection (Morimoto et al. 1995). In both models, C/EBPalpha and DBP mRNA levels in the liver are lower, whereas C/EBPbeta mRNA is higher prior to a low albumin mRNA level. Mueller (1992) reported that low albumin gene expression in regenerating rat liver after carbon tetrachloride administration is associated with a decrease in the binding activity of the liver nuclear extract to B and D sites of the albumin promoter identified by gel mobility shift analysis. Mueller (1992) also showed that C/EBPalpha and DBP are lower in the regenerating liver, which is consistent with our previous results.

The results of the present study showed that, in contrast to other hypoalbuminemia models, such as those involving regenerating liver and inflammation, there were no significant differences in the binding activities of liver nuclear extracts to the B and D sites of the albumin promoter between the protein-deprived rats and the pair-fed controls. To further analyze the nature of the protein bound to the D site oligomer, we subjected the membrane filter of the gel shift assay to Western blot analysis using antibodies against the D site binding transcription factors. Again, however, no difference was observed in the protein levels of C/EBPalpha , C/EBPbeta and DBP between the two groups. Western blot analysis showed no significant difference in LAP protein abundance between the two groups. However, because LIP could not be detected with the assay system we used, the LAP/LIP ratio is not known. Hepatocyte nuclear factor-1 mRNA also did not significantly differ between the two groups, in agreement with the gel-shift analysis of the B site.

We thus came to the conclusion that the albumin promoter and its binding proteins, C/EBPalpha , C/EBPbeta , HNF-1 and DBP, are unlikely to play major roles in the down-regulation of albumin gene transcription in protein deprivation. Therefore, a different regulatory mechanism must be involved in the down-regulation of albumin gene transcription in the liver of protein-deprived rats.

One possibility is that an enhancer element plays an important role in protein-energy malnutrition, because the albumin gene has an enhancer element far upstream (-10.4 to 8.5 kb) (Pinkert et al. 1987), consisting of several sites where other nuclear factors bind in vitro. Of these sites, those designated eE, eG and eH were found to be essential for the enhancer function (Liu et al. 1991). The eE site binds C/EBPalpha and C/EBPbeta (Johnson et al. 1987), the eG site binds liver-enriched proteins of the HNF-3 family, and the eH site binds both HNF-3 and the factor NF1 (Jackson et al. 1993). Further experiments, such as analysis of far upstream enhancer elements of the albumin gene, should be performed to clarify the mechanism of hypoalbuminemia under protein deprivation.

Another possibility is that transcriptional regulation may not play a major role in down-regulation of albumin synthesis during protein deprivation because posttranscriptional regulation is more important. The amount of translatable mRNA is associated with synthesis, degradation and intracellular distribution of mRNA. This study indicated that the decrease in albumin mRNA may be regulated at the level of mRNA degradation. During starvation, albumin synthesis decreases because albumin mRNA is stored in the messenger ribonucleoprotein fraction that is untranslated (Yap et al. 1978). In this study, we did not examine the cytoplasmic distribution of albumin mRNA during protein deprivation. Also, Perozzi et al. (1989) reported that in protein-deficient rats albumin mRNA is present in lower amounts, and a small but consistent fraction of mRNA is also present in the nonpolysomal fractions that are not translated. However, this phenomenon does not seem to be specific to albumin mRNA under protein deficiency, because it was seen in all mRNAs they examined.

In conclusion, liver-specific nuclear transcription factors for the proximal promoter do not seem to play a major role in down-regulation of the albumin gene in protein deficiency.

ACKNOWLEDGMENT

We thank U. Schibler for kindly providing the anti-DBP antibody.

FOOTNOTES

1   Presented in part at the European Society of Parenteral and Enteral Nutrition, September 1996, Geneva, and published in abstract form [Ogawa, A., Yano, M., Tsujinaka, T., Ebisui, C., Morimoto, T., Kishibuchi, M., Morita, S., Taniguchi, M., Shiozaki, H., & Monden, M. (1996) Role of nuclear transcription factors in regulation of albumin gene in protein-deprived rat liver. Clin. Nutr. 15(suppl): 2.]
2   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3   To whom correspondence and reprint requests should be addressed.
4   Abbreviations used: C/EBP, CCAAT/enhancer binding protein; DBP, D site binding protein; HNF, hepatocyte nuclear factor; LAP, liver-enriched transcriptional activation protein; LIP, liver-enriched transcriptional inhibitory protein; SSC, saline-sodium citrate; RBP, retinol-binding protein.

Manuscript received 22 July 1996. Initial reviews completed 19 August 1996. Revision accepted 2 April 1997.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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