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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2131-2136, July 2003

Nutrient-Gene Interactions

Vitamin A Depletion Is Associated with Low Phosphoenolpyruvate Carboxykinase mRNA Levels during Late Fetal Development and at Birth in Mice

Saheli Ghoshal, Saritha Pasham, Daniel P. Odom, Harold C. Furr and Mary M. McGrane2

Department of Nutritional Sciences and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269

2To whom correspondence should be addressed. E-mail: mary.mcgrane{at}uconn.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is repressed during fetal liver development and activated at birth. It has been shown that the PEPCK gene is a retinoid-responsive gene, but whether it is regulated by vitamin A in the fetus has not been established. In this study, we found that PEPCK mRNA can be detected in the murine fetal liver as early as gestational d 17. In addition, expression and cAMP induction of the PEPCK gene during late gestation and at birth require vitamin A sufficiency in the fetus and neonate. The PEPCK promoter contains several regulatory elements that bind a diverse array of transcription factors and nuclear coregulators, although it is largely unknown which of these factors are expressed early in liver development. Expression of some of these nuclear factors in livers of fetal mice was investigated by immunohistochemistry (IHC). Fetuses were from dams that were fed from the beginning of gestation diets that were adequate or devoid of vitamin A. Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) was expressed at the earliest stage of liver development on d 11, whereas retinoid X receptor {alpha} (RXR{alpha}) and nuclear coactivator CREB-binding protein (CBP) were expressed from d 16 onward. Although expressions of RXR{alpha} and CBP in livers of vitamin A–sufficient and vitamin A–depleted fetal mice did not differ, the level of HNF4{alpha} was consistently lower in the latter. Our findings strongly suggest that vitamin A is required during liver development for staged expression of the PEPCK gene and that HNF4{alpha} may be involved in mediating vitamin A regulation of the PEPCK gene at these critical periods.

KEY WORDS: • phosphoenolpyruvate carboxykinase • vitamin A • development • hepatocyte nuclear factor 4{alpha} • retinoic acid response element

Vitamin A is required for normal embryonic and fetal development, as well as maintenance of the fully differentiated state in the adult (1). Although limited data exist in humans, complete maternal vitamin A deficiency during gestation in rats leads to a complicated array of disorders including infertility, spontaneous abortion, fetal malformation and late fetal death (2–5). For the most part, however, these disorders are the result of severe vitamin A deficiency in the dam. Subclinical deficiency is much more prevalent in the developing world (6) and is recognized as a major public health problem with increased risk of fetal and infant mortality and morbidity (7). However, the effect of maternal subclinical vitamin A deficiency on regulation of fetal growth and organ development during the stages of gestation has not been examined, nor has the effect of moderate vitamin A deficiency on gene expression in developing tissues received much attention. In addition, there is a notable lack of information on the effect of either severe or moderate vitamin A deficiency on the developing liver, in part due to the fact that the critical period for liver organogenesis is relatively late (mid-gestation). In this study, we began to investigate the effect of vitamin A depletion on the developing liver by examining the phosphoenolpyruvate carboxykinase (PEPCK) gene as a prototype retinoid-responsive gene. The PEPCK gene is a particularly useful prototype for examining liver development because it is inhibited until late gestation and activated at birth (8,9). PEPCK is the last gluconeogenic enzyme to be expressed in liver development and is rate limiting in the liver’s capacity for gluconeogenesis at this time.

All-trans retinoic acid (at-RA) and its 9-cis isomer are the active metabolites of vitamin A in the nucleus of the cell. These retinoids bind to high affinity nuclear receptors, i.e., the retinoic acid receptors (RAR) and retinoid X receptors (RXR), which act as transcription factors that control expression of specific genes. The gene encoding a key regulatory enzyme in hepatic gluconeogenesis, PEPCK, is one of the target genes of both at- and 9-cis RA (10). Extensive studies on the PEPCK gene have shown that the PEPCK promoter contains three retinoic acid response elements (RARE1, RARE2 and RARE3) (11–15), and at-RA treatment induces PEPCK gene transcription in H4IIE hepatoma cells (12–14). Conducting in vivo studies with transgenic mice, our laboratory has shown that vitamin A deficiency significantly decreases hepatic PEPCK transgene and endogenous PEPCK mRNA levels in adult mice (10,16). Furthermore, PEPCK transgenes are differentially responsive to treatment with at-RA or 9-cis RA (10). Overall, regulation of the PEPCK gene by retinoids is now well characterized in adult liver; however, there is no information concerning vitamin A regulation of PEPCK gene expression in the developing fetal liver. This is partly because PEPCK mRNA is difficult to measure in fetal liver in which expression is low and initiated in late gestation. During gestation, the fetus is nourished by the maternal glucose supply, and PEPCK activity is not detected in fetal liver until the perinatal period. Hepatic PEPCK gene transcription is significantly increased at birth due to the decrease in the insulin/glucagon ratio that occurs at this time, although expression can be induced in fetal liver by cAMP treatment (17–21). Hepatic gluconeogenesis is initiated a few hours after birth and maintains glucose homeostasis in neonates. Given the critical role of vitamin A in cellular differentiation, we sought to determine the effect of vitamin A depletion on the stages of metabolic differentiation that occur in the developing liver in late gestation, assessed by PEPCK gene expression.

Recently, we identified specific nuclear receptors present in mouse liver nuclear extract that bind PEPCK RARE (22). Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is the major nuclear receptor that binds PEPCK RARE1; RXR{alpha} binds PEPCK RARE1, RARE2 and RARE3; and RAR{alpha} binds PEPCK RARE2 (22). In addition, the chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) also binds RARE1 and RARE2, most likely as an inhibitory transcription factor. RXR{alpha} has been shown to be involved in liver organogenesis and mouse fetuses homozygous for a mutant RXR{alpha} die between d 12 and 16 of gestation (23,24). The defects in these fetuses resemble those with fetal vitamin A deficiency syndromes. HNF4{alpha} is another highly conserved member of the nuclear receptor superfamily, critical for development, liver cell differentiation and liver-specific gene expression (25–27). However, to date, nuclear receptor expression in the developing liver has not been fully characterized despite the central role of these transcription factors in regulating liver-specific genes. Therefore, we sought to determine whether nuclear receptors that bind PEPCK RARE1, RARE2, and RARE3 are present in the developing liver, and therefore available to regulate fetal PEPCK gene expression. Further, we determined the gestational time of onset of expression of the nuclear receptor genes, to correlate their induction with the onset of PEPCK gene expression late in gestation. The time of onset of RXR{alpha} and CREB-binding protein (CBP) expression on gestation d 16 is just before the earliest time of PEPCK mRNA appearance on gestation d 17. The effect of vitamin A depletion was subsequently determined and provides evidence that HNF4{alpha} expression is regulated by vitamin A in fetal liver.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Animals and diet.

For experiments with both transgenic and nontransgenic mice, C57BL/6XSJL hybrid mice (Jackson Laboratory, Bar Harbor, ME) were used. Two lines of transgenic mice, PEPCK (460) and PEPCK (355), were used. These mice were produced as described by McGrane et al. (28) using standard microinjection procedures. In transgenic mouse lines, PEPCK DNA segments from –460 to +73 or –355 to +73 were ligated to a region of the bovine growth hormone (bGH) marker gene. The long half-life of the PEPCK-bGH mRNA enables sensitive detection of low levels of mRNA in fetal liver. Mice were housed in stainless steel cages at 21°C with a 12-h light:dark cycle. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Connecticut.

Fetuses were made vitamin A–depleted (VAD) by feeding females the AIN 76A diet without vitamin A (29) beginning on d 0 of gestation. Successful mating was determined by the presence of a vaginal plug and the day of appearance was designated d 0. Vitamin A–sufficient (VAS) mice were fed the same diet with 3600 retinol equivalents of retinyl esters/kg diet (Dyets, Bethlehem, PA).

Animal treatment.

For experiments with fetuses at d 17 of gestation, pregnant mice were anesthetized and the uteruses exposed by partial laparotomy. In one uterine horn, the fetuses were injected intraperitoneally with dibutyryl cAMP (Bt2cAMP) and theophylline (both 30 mg/kg body) through the uterine wall. In the other uterine horn, fetuses were injected intraperitoneally with saline alone. Immediately upon termination of injections, the maternal incision was closed. At the designated time after treatment, individual livers from each fetus from the treated uterine horn were removed for RNA extraction, as were individual fetal livers from the control uterine horn group. This was repeated in 3–4 dams for each treatment. For experiments in the postnatal period, pups were injected with either Bt2cAMP and theophylline at the above dose or with saline alone. Individual livers were excised from each neonate for RNA extraction and Northern blot analysis. In both groups, mice were killed 1 h after treatment.

Collection of fetuses for RNA analysis or for histologic examination.

At d 11, 13, and 16–19 of gestation, pregnant females (n = 4–5) were killed and fetuses removed from the uterus; d 11 fetuses were immediately placed in 10% neutral buffered formalin for immunohistochemistry (IHC). At later days of gestation, fetal livers were removed and either frozen immediately for subsequent RNA analysis or placed in 10% neutral buffered formalin for IHC. For experiments in the postnatal period, pups were killed by decapitation and livers removed and treated similarly to fetal livers. Individual liver samples were used for RNA analysis. For IHC, liver samples from four fetuses or three pups were used per time point.

Analysis of the retinol concentration of fetal livers.

Liver samples were pooled from four fetuses per group and saponified in methanolic KOH overnight at room temperature as described by Furr and colleagues (30). Each sample was reconstituted in 250 µL of 2-propanol/dichloroethane (80:20), and 25-µL aliquots were analyzed by reversed-phase HPLC. Quantitative analysis of retinol was carried out on a Microsorb C18 column (Varian, Palo Alto, CA), with a mobile phase of ethanol/water (95:5, v/v) at a flow rate of 1.0 mL/min, with absorbance detection at 325 nm. External standardization was used, with correction for recovery of the internal standard. Total vitamin A (as retinol) was calculated per unit weight of liver sample.

RNA isolation and Northern blot analysis.

Total RNA from whole fetal livers (30–100 mg) was extracted using Trizol reagent (Life Technologies, Carlsbad, CA). Northern blot analysis was performed as described previously (10). Membranes were exposed to Kodak (Rochester, NY) Biomax film at -80°C for 2–3 d (PEPCK mRNA) or 6–12 h (rpl 32 mRNA). The cDNA probes used were a 1.6-kb BglII fragment from the rat PEPCK cDNA (31); a 1.2-kb EcoRI-BamHI fragment from the bovine growth hormone (bGH) cDNA; and a 1.6-kb SstI fragment from the mouse rpl 32 cDNA (32). The latter was used as a control for total RNA per lane.

Immunohistochemistry (IHC).

For IHC, the following polyclonal antibodies were used: (i) goat anti-HNF4{alpha}, (ii) rabbit anti-RXR{alpha}, and (iii) goat anti-CBP (Santa Cruz Biotechnology, Santa Cruz, CA). IHC was carried out on 5 µm-thick sections of paraffin-fixed tissues by the streptavidin-biotin immunoperoxidase method. The deparaffinized and rehydrated sections were incubated overnight in a humidified chamber at 4°C in a 1:100 dilution of primary antibody. After rinsing in PBS for 15 min, sections were incubated for 1 h at 1:400 dilution of biotinylated anti-rabbit or anti-goat secondary antibody, as appropriate. Liver sections were washed again with PBS and incubated with ABC reagent (Vector Laboratories, Burlingame, CA) containing horseradish peroxidase. Immunoreactivity was visualized using diaminobenzidine tetrachydrochloride. The preparations were lightly counterstained with eosin and mounted. Photographs were taken using an Olympus IX70 microscope (Olympus America, Melville, NY).

Statistical analysis.

Results of HPLC analysis of retinol concentrations and Northern blot analysis of mRNA levels were reported as means ± SEM. Differences between group means were analyzed by Student’s t tests. Differences were considered significant at P < 0.05. All data were evaluated using SPSS version 9 (SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
We measured hepatic PEPCK transgene expression at gestation d 15, 17 and 19, and d 2 after birth to determine the earliest time of PEPCK gene transcription in fetal liver and the subsequent pattern of expression (Fig. 1). Although no PEPCK transgene mRNA was detected on d 15 (data not shown), PEPCK transgene mRNA was detected as early as d 17. By d 19, PEPCK transgene mRNA levels were twofold greater than those measured on d 17. After birth, on postnatal d 2, PEPCK mRNA levels were ~60-fold greater than those measured on d 17.



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  		FIGURE 1 Phosphoenolpyruvate carboxykinase (PEPCK) transgene expression during late gestation and in the postnatal period in livers of mice whose dams were vitamin A–sufficient (VAS) or –depleted (VAD). (Upper panel) Autoradiogram of representative Northern blot. Total RNA was extracted from whole fetal livers (d 17 and 19) and from livers of 2-d-old mice. Individual liver RNAs (15 µg/lane) were analyzed by Northern blot. The same blot was hybridized with a 32P-labeled rpl32 probe to correct for variation in RNA loading per lane. (Lower panel) PEPCK transgene mRNA levels were determined by densitometric scan of representative autoradiograms. Each bar is the mean (n = 4).

 
To determine the effects of vitamin A on the developmental pattern of PEPCK gene expression, we first measured the retinol concentration in fetal livers of pregnant dams fed VAS and VAD diets, to ensure that the fetal levels reflected the maternal diet. Retinol concentrations were measured in whole embryos on d 11, and in fetal livers on d 13 and 17 of gestation. Although there was only a marginal reduction in retinol concentration in d 11 VAD embryos, there were reductions of 24 and 42% in retinol level in fetal livers on d 13 (P = 0.08) and d 17 (P < 0.004), respectively, compared with VAS fetuses (Table 1). In the experiments described here, there were no differences in length of gestation, fetal weights, or number of live neonates between the VAD and VAS groups (data not shown).


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  		TABLE 1 Retinol concentrations of vitamin A-sufficient (VAS) and vitamin A-depleted (VAD) fetal mice on gestational d 11, 13 and 171

 
To determine the effect of vitamin A depletion on fetal and postnatal PEPCK mRNA levels, we measured endogenous PEPCK and PEPCK transgene mRNAs. Gestation d 17 is the earliest time we detected endogenous PEPCK or PEPCK transgene mRNA in fetal liver by Northern blot analysis. At d 17 of gestation, in vitamin A–deficient PEPCK (355) and PEPCK (460) mice, the mRNA levels were 70 and 50% lower, respectively, than in control transgenic mice (P < 0.005) (Fig. 2). Endogenous PEPCK mRNA levels were also measured on d 17. There was a 70% reduction of PEPCK mRNA in VAD fetuses compared with the control VAS fetuses (data not shown). Therefore, vitamin A deficiency has a significant effect on the earliest phase of expression of the PEPCK gene. Similarly, in the postnatal period, there were 70 and 50% decreases in PEPCK transgene mRNA levels in VAD PEPCK (355) and PEPCK (460) mice, respectively, compared with control pups (P < 0.005) (Fig. 2). Thus, the normal induction of the hepatic PEPCK gene after birth in response to the decrease in the insulin/glucagon ratio is significantly inhibited by VAD during gestation.



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  		FIGURE 2 Effect of vitamin A status on phosphoenolpyruvate carboxykinase (PEPCK) transgene mRNA levels on fetal d 17 and postnatal d 2 in livers of mice whose dams were vitamin A–sufficient (VAS) or –depleted (VAD). (A) Autoradiograms of representative Northern blots. Total RNA was obtained from VAS and VAD fetuses and pups and processed and analyzed by Northern blot as described in Figure 1. (B) PEPCK transgene mRNA levels were determined by densitometric scan of representative autoradiograms for VAS and VAD mice at two different time points and normalized to rpl32 mRNA. Each bar is the mean ± SEM (n = 4 for fetal d 17 and n = 5 for postnatal d 2). *Different from VAS (P < 0.005).

 
The observation that PEPCK gene expression at birth is significantly attenuated by VAD led us to hypothesize that vitamin A is required for glucagon/cAMP induction of the PEPCK gene. The vitamin A requirement for cAMP induction of the PEPCK gene was tested in late gestation. In VAS and VAD fetuses on gestation d 17, in utero Bt2cAMP treatment induced PEPCK mRNA levels compared with saline-treated controls (P < 0.007 for VAS and P < 0.05 for VAD) (Fig. 3A and B). However, Bt2cAMP stimulation of PEPCK mRNA levels in VAD fetuses was less than that in VAS fetuses (P < 0.008). Bt2cAMP treatment increased PEPCK mRNA levels sevenfold in VAS fetuses, but only 3.2-fold in VAD fetuses compared with saline-treated controls. Next, we determined the ability of cAMP to induce PEPCK mRNA levels in VAS and VAD pups in the postnatal period. Notably, cAMP induction of PEPCK mRNA levels was 2.2-fold greater in the VAS group than in the VAD group on postnatal d 2 (Fig 3C).



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  		FIGURE 3 Effect of vitamin A status on cAMP response of the phosphoenolpyruvate carboxykinase (PEPCK) gene on fetal d 17 and postnatal d 2 in livers of mice whose dams were vitamin A–sufficient (VAS) or –depleted (VAD). (A) Autoradiogram of representative Northern blot. On d 17, VAS and VAD fetuses were injected with either cAMP or saline as described in Materials and Methods. Total RNA was obtained and processed and analyzed by Northern blot as described in Figure 1. The endogenous PEPCK mRNA of 2.8 kb is shown in the figure. (B) PEPCK mRNA levels were determined by densitometric scan of representative autoradiograms for cAMP-treated and saline-treated VAS and VAD mice on fetal d 17. Hepatic PEPCK mRNA levels in untreated (saline) VAS and VAD fetuses are set at an arbitrary value of 1.0 and the relative change in respective mRNA levels in cAMP-treated VAS and VAD fetuses are indicated. Each bar is the mean ± SEM (n = 4). *Different from saline-treated controls (P < 0.007 for VAS and P < 0.05 for VAD); **different from VAS cAMP-treated (P < 0.008). (C) PEPCK mRNA levels were determined by densitometric scan of representative autoradiograms for cAMP-treated and saline-treated VAS and VAD mice on postnatal d 2 as described in (B). Each bar is the mean ± SEM (n = 4). *Different from saline treated controls (P < 0.001); **different from VAS cAMP-treated (P < 0.01).

 
The PEPCK promoter contains several regulatory elements that are bound by a diverse array of transcription factors and nuclear coregulators, although it is not clear which of these factors are expressed in fetal liver. We performed IHC on fetal liver on gestation days 11, 13, 16 and 18 to identify the developmental expression pattern of nuclear receptors and associated coregulators that bind the PEPCK RARE (Fig. 4). We found that HNF4{alpha} was expressed very early in liver development, i.e., on d 11 in the hepatic primordium. RXR{alpha} and the coactivator CBP were expressed from d 16 onward. RAR{alpha}, RARß and COUP-TFII were not detected in fetal liver on the days examined, nor were nuclear coregulators SRC-1, SMRT and NCo-R (data not shown).



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  		FIGURE 4 Immunohistochemical (IHC) localization of hepatocyte nuclear factor 4{alpha} (HNF4{alpha}), retinoid X receptor {alpha} (RXR{alpha}) and CREB binding protein (CBP) in livers of fetal mice whose dams were vitamin A–sufficient (VAS) or –depleted (VAD) at d 11, 13, 16 and 18 of gestation. On d 11, IHC was performed on sagittal sections of whole embryo (magnification X100, inset magnification X400). On d 13, 16 and 18, fetal livers were isolated and IHC was performed (magnification X400). Arrows point to the respective nuclear proteins.

 
Finally, we examined the effect of VAD on the developmental expression pattern of HNF4{alpha}, RXR{alpha} and CBP in fetal liver by IHC. HNF4{alpha} was consistently lower in VAD fetal livers on gestation d 13, 16 and 18 (Fig. 5). By comparison, there were no apparent differences in the pattern of RXR{alpha} and CBP in VAD and VAS fetal livers (data not shown).



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  		FIGURE 5 Immunohistochemical (IHC) localization of hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) at d 13, 16 and 18 of gestation in livers of fetal mice whose dams were vitamin A–sufficient (VAS) or –depleted (VAD)(magnification X400). Arrows point to the respective nuclear proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Severe vitamin A deficiency during pregnancy may result in termination of pregnancy and fetal death (2). Therefore, to determine the effect of vitamin A deprivation on hepatic gene expression during fetal development, we made mouse fetuses vitamin A depleted, but provided enough vitamin A to support gestation. We achieved this by initiating the vitamin A–deficient diet on d 0 of pregnancy. This approach did not require retinol supplementation to prevent resorption of the fetuses. Therefore, a relatively short period of VAD diet treatment, initiated at the onset of pregnancy, resulted in a depletion of fetal liver retinol at mid- and late gestation. Furthermore, the measured vitamin A depletion in the fetal livers indicated that maternal vitamin A stores were not sufficient to supply adequate retinol to the fetus when dietary vitamin A was removed during pregnancy.

The liver goes through a number of defined developmental stages during gestation. In mice, the final stage is initiated on fetal d 17 when liver cells undergo a transformation from primarily hematopoietic support cells to fully differentiated hepatocytes. In the last phase of liver development, a number of metabolic genes, under the control of regulatory transcription factors, are induced (33). Additionally, hepatocytes develop cell junctions and begin to form the polarized epithelium characteristic of the adult liver (34). It is during this stage in fetal liver that the cytosolic PEPCK gene attains the capacity to be expressed. PEPCK is the last gluconeogenic gene to be activated in the developing liver; therefore, PEPCK gene expression is the determining factor in the gluconeogenic capacity of the liver at this stage. PEPCK mRNA has been detected at low levels on d 19 in mouse liver (21) and is inducible at d 17 by cAMP (19). Therefore, the PEPCK gene acquires competence to be activated at the beginning of the last phase of liver development. Vitamin A in the form of at-RA is a local signaling molecule that potentially contributes to terminal hepatocyte differentiation. at-RA is required for expression of the PEPCK gene in adult liver, which contributes to the maintenance of the fully differentiated metabolic state of the mature hepatocyte. Therefore, given its importance in cellular differentiation, at-RA potentially determines the transition to competence of the PEPCK gene in the final stage of hepatocyte differentiation in fetal liver. The PEPCK gene serves as a prototype gene for late fetal development because at-RA may activate a series of metabolic genes and transcription factors at this stage in liver ontogeny.

We investigated the requirement for vitamin A in induction of the PEPCK gene during late gestation, assessed by the measurement of PEPCK mRNA levels in fetal liver. PEPCK activity is regulated primarily at the level of gene transcription and mRNA concentration, not at the level of translation or post-translational modification (35). Therefore, measurement of PEPCK mRNA is generally accepted as an accurate determinant of PEPCK enzyme concentration and activity. Relative to vitamin A status in the developing liver, Goodman and colleagues described three phases of vitamin A accumulation during fetal development in the rat: an early phase (d 7–9) in which there is substantial accumulation of retinol; a second phase (d 11–14) in which retinol and retinol binding protein accumulate; and a third phase (d 16–20) in which transplacental transport of retinol is extensive and highly regulated (3,4). In the last phase, there is a large increase in fetal whole-body vitamin A. Therefore, retinol levels increase in the fetal liver just before the time of increased metabolic gene expression and alterations in hepatocyte morphology. The transition to competence of the PEPCK gene may be induced by the increase in fetal liver retinol, assuming a concomitant increase in at-RA. Inadequate retinol, as would occur with a vitamin A–deficient diet, and inadequate at-RA availability may delay or inhibit this transition. Our results show that vitamin A depletion significantly decreases PEPCK gene expression and inhibits induction by cAMP during late gestation as well as in the immediate postnatal period. Therefore, retinol (presumably at-RA in the nucleus) is a dominant positive regulator of PEPCK gene expression in the perinatal period.

Liver-specific HNF4{alpha} is a member of the nuclear receptor superfamily of transcription factors. It has been reported that the transition to the final phase of liver differentiation requires expression of the HNF4{alpha} gene (27,36), as do earlier phases of embryonic development (37). In the absence of HNF4{alpha}, the genes for apolipoproteins, serum factors, downstream transcription factors and metabolic enzymes are not induced in the maturing hepatocyte (38). Of particular interest during development is that HNF1{alpha} is also a target gene of HNF4{alpha}; together these nuclear receptors play a crucial role in liver cell differentiation (25). HNF4{alpha}, then, is potentially a key transcription factor involved in eliciting the terminal phase of hepatocyte differentiation (27). Consistent with this, when the HNF4{alpha} gene is transfected into dedifferentiated hepatoma cells, these cells assume the epithelial morphology of fully differentiated hepatocytes (38). Relative to the PEPCK gene and the potential role of HNF4{alpha} in the acquisition of competence, we showed by chromatin immunoprecipitation that HNF4{alpha} binds the PEPCK retinoic acid response element (RARE) 1 element in liver in vivo (Scribner, K. and McGraw, M., unpublished results). To date, however, it is not clear what signal directs the association of HNF4{alpha} with the PEPCK RARE1. The findings reported here show that HNF4{alpha} is one of the first nuclear receptors to be expressed in the developing liver and that vitamin A depletion decreases the levels of nuclear HNF4{alpha} in the developing mouse liver from d 13 to 18. Thus, during liver development, reduced expression of the HNF4{alpha} gene due to VAD and the concomitant decrease in expression of the HNF1{alpha} gene together may lead to inadequate metabolic differentiation of liver cells, as exhibited by decreased expression (and hormonal responsiveness) of the PEPCK gene. Therefore, we predict that the HNF4{alpha} gene is responsive to retinoids in the fetal liver and that HNF4{alpha} binding to the PEPCK RARE1 determines expression of the PEPCK gene, as a prototypical late phase gene, in fetal liver in response to vitamin A status of the mother.


   FOOTNOTES
 
1 Supported by U.S. Department of Agriculture grant 9900685 (to M.M.M.). Back

3 Abbreviations used: at-RA, all-trans retinoic acid; bGH, bovine growth hormone; Bt2cAMP, dibutyryl cAMP; CBP, CREB binding protein; COUP-TFII, chicken ovalbumin upstream promoter transcription factor II; HNF4{alpha}, hepatocyte nuclear factor {alpha}; IHC, immunohistochemistry; PEPCK, phosphoenolpyruvate carboxykinase; RARE, retinoic acid response element; RXR{alpha}, retinoid X receptor {alpha}; VAD, vitamin A depleted; VAS, vitamin A sufficient. Back

Manuscript received 16 January 2003. Initial review completed 7 February 2003. Revision accepted 2 April 2003.


   LITERATURE CITED
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Ross, S. A., McCaffery, P. J., Drager, U. C. & DeLuca, L. M. (2000) Retinoids in embryonal development. Physiol. Rev. 80:1021-1054.[Abstract/Free Full Text]

2. Thompson, J. N., Howell, J. M. & Pitt, G.A.J. (1964) Vitamin A and reproduction in rats. Proc. R. Soc. B 159:510-535.[Medline]

3. Takahashi, Y. I., Smith, E. S., Winick, M. & Goodman, D. S. (1975) Vitamin A deficiency and fetal growth and development in the rat. J. Nutr. 105:1299-1310.

4. Takahashi, Y. I., Smith, E. S. & Goodman, D. S. (1977) Vitamin A and retinol-binding protein metabolism during fetal development in the rat. Am. J. Physiol. 233:E263-E272.

5. Antipatis, C., Grant, G. & Ashworth, C. J. (2000) Moderate maternal vitamin A deficiency affects perinatal organ growth and development in rats. Br. J. Nutr. 84:125-132.[Medline]

6. Rahmathullah, L., Underwood, B. A., Thulasiraj, R. D., Milton, R. C., Ramaswamy, K., Rahmathullah, R. & Babu, G. (1990) Reduced mortality among children in southern India receiving a small weekly dose of vitamin A. N. Engl. J. Med. 323:929-935.[Abstract]

7. Underwood, B. A. & Arthur, P. (1996) The contribution of vitamin A to public health. FASEB J. 10:1040-1048.[Abstract]

8. Ballard, F. J. (1971) The development of gluconeogenesis in rat liver.Controlling factors in the newborn. Biochem. J. 124:265-274.[Medline]

9. Callikan, S. & Girard, J. (1979) Perinatal development of gluconeogenic enzymes in rabbit liver. Biol. Neonate 36:78-84.[Medline]

10. Shin, D. J. & McGrane, M. M. (1997) Vitamin A regulates genes involved in hepatic gluconeogenesis in mice: phosphoenolpyruvate carboxykinase, fructose-1, 6-bisphosphatase and 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase. J. Nutr. 127:1274-1278.[Abstract/Free Full Text]

11. Lucas, P. C., O’Brien, R. M., Mitchell, J. A., Davis, C. M., Imai, E., Forman, B. M., Samuels, H. H. & Granner, D. K. (1991) A retinoic acid response element is part of a pleiotropic domain in the phosphoenolpyruvate carboxykinase gene. Proc. Natl. Acad. Sci. U.S.A. 88:2184-2188.[Abstract/Free Full Text]

12. Lucas, P. C., Forman, B. M., Samuels, H. H. & Granner, D. K. (1991) Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding. Mol. Cell. Biol. 11:5164-5170.[Abstract/Free Full Text]

13. Hall, R. K., Scott, D. K., Noisin, E. L., Lucas, P. C. & Granner, D. K. (1992) Activation of the phosphoenolpyruvate carboxykinase gene retinoic acid response element is dependent on a retinoic acid receptor/coregulator complex. Mol. Cell. Biol. 12:5527-5535.[Abstract/Free Full Text]

14. Scott, D. K., Mitchell, J. & Granner, D. K. (1996) Identification and characterization of the second retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter. J. Biol. Chem. 271:6260-6264.[Abstract/Free Full Text]

15. Devine, J. H., Eubank, D. W., Clouthier, D. E., Tontonoz, P., Spiegelman, B. M., Hammer, R. E. & Beale, E. G. (1999) Adipose expression of the phosphoenolpyruvate carboxykinase promoter requires peroxisome proliferator-activated receptor gamma and 9-cis-retinoic acid receptor binding to an adipocyte-specific enhancer in vivo. J. Biol. Chem. 274:13604-13612.[Abstract/Free Full Text]

16. Shin, D. J., Tao, A. & McGrane, M. M. (1995) Effects of vitamin A deficiency and retinoic acid treatment on expression of a phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice. Biochem. Biophys. Res. Commun. 213:706-714.[Medline]

17. Hanson, R. W. & Ballard, F. J. (1975) Hormonal regulation of hepatic P-enolpyruvate carboxykinase (GTP) during development. Fed. Proc. 34:166-171.[Medline]

18. Garcia-Ruiz, J. P., Ingram, R. & Hanson, R. W. (1978) Changes in hepatic messenger RNA for phosphoenolpyruvate carboxykinase (GTP) during development. Proc. Natl. Acad. Sci. U.S.A. 75:4189-4193.[Abstract/Free Full Text]

19. Lamers, W. H., Hanson, R. W. & Meisner, H. M. (1982) cAMP stimulates transcription of the gene for cytosolic PEPCK in rat liver nuclei. Proc. Natl. Acad. Sci. U.S.A. 79:5137-5141.[Abstract/Free Full Text]

20. Mencher, D., Cohen, H., Benvinisty, N., Meyuhas, O. & Reshef, L. (1984) Primary activation of cytosolic phosphoenolpyruvate carboxykinase gene in fetal rat liver and the biogenesis of its mRNA. Eur. J. Biochem. 141:199-203.[Medline]

21. McGrane, M. M., Yun, J. S., Moorman, A.F.M., Lamers, W. H., Hendrick, G. K., Arafah, B. M., Park, E. A., Wagner, T. E. & Hanson, R. W. (1990) Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice. J. Biol. Chem. 265:22371-22379.[Abstract/Free Full Text]

22. Shin, D. J., Odom, D. P., Scribner, K., Ghoshal, S. & McGrane, M. M. (2002) Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver. Mol. Cell. Endocrinol. 195:39-54.[Medline]

23. Kastner, P., Leid, M. & Chambon, P. (1994) Role of nuclear retinoic acid receptors in the regulation of gene expression. Blomhoff, R. eds. Vitamin A in Health and Disease 1994:189-238 Marcel Dekker New York, NY. .

24. Sucov, H. M., Dyson, E., Gumeringer, C. L., Price, J., Chien, K. R. & Evans, R. M. (1994) RXR alpha mutant mice establish a genetic basis for vitamin A signaling in heart morphogenesis. Genes Dev. 8:1007-1018.[Abstract/Free Full Text]

25. Duncan, S., Manova, K., Chen, W., Hoodless, P., Weinstein, D. C., Bachvarova, R. F. & Darnell, J. E., Jr (1994) Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst. Proc. Natl. Acad. Sci. U.S.A. 91:7598-7602.[Abstract/Free Full Text]

26. Chen, D., Park, Y. & Kemper, B. (1994) Differential protein binding and transcriptional activities of HNF-4 elements in three closely related CYP2C genes. DNA Cell. Biol. 13:771-779.[Medline]

27. Li, J., Ning, G. & Duncan, S. A. (2000) Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha. Genes Dev. 14:464-474.[Abstract/Free Full Text]

28. McGrane, M. M., de Vente, J, Yun, J., Bloom, J., Park, E., Wynshaw-Boris, , Wagner, T., Rottman, F. M. & Hanson, R. W. (1988) Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice. J. Biol. Chem. 263:11443-11451.[Abstract/Free Full Text]

29. Bieri, J. G., Stoewsand, G. S., Briggs, G. M., Philips, R. W., Woodard, J. C. & Knapka, J. J. (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.

30. Furr, H. C. (1990) Reversed phase high-performance liquid chromatography of retinyl esters. Methods Enzymol. 189:85-94.[Medline]

31. Yoo-Warren, H., Monahan, J. E., Short, J., Short, H., Bruzel, A., Wynshaw-Boris, A., Meisner, H. M., Samols, D. & Hanson, R. W. (1983) Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat. Proc. Natl. Acad. Sci. U.S.A. 80:3656-3660.[Abstract/Free Full Text]

32. Jacks, C. M., Powaser, C. B. & Hackett, P. B. (1989) Sequence analysis of a processed mouse gene coding for ribosomal protein L32. Gene 74:565-570.

33. Wang, N. D., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R. & Darlington, G. J. (1995) Impaired energy homeostasis in C/EBP alpha knockout mice. Science (Washington, DC) 269:1108-1112.[Abstract/Free Full Text]

34. Stamaloglou, S. C. & Hughes, R. C. (1994) Cell adhesion molecules in liver function and pattern formation. FASEB J. 8:420-427.[Abstract]

35. Hanson, R. W. & Reshef, L. (1997) Regulation of phosphoenolpyruvate carboxykinase gene expression. Annu. Rev. Biochem. 66:581-611.[Medline]

36. Duncan, S. A., Nagy, A. & Chan, W. (1997) Murine gastrulation requires HNF4 regulated gene expression in the visceral endoderm: tetraploid rescue of HNF4(-/-) embryos. Development 124:279-287.[Abstract]

37. Chen, W. S., Manova, K., Weinstein, D. C., Duncan, S. A., Plump, A. S., Prezioso, V. R., Bachvarova, R. F. & Darnell, J. E., Jr (1994) Disruption of the HNF4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos. Genes Dev. 8:2466-2477.[Abstract/Free Full Text]

38. Spath, G. F. & Weiss, M. C. (1998) Hepatocyte nuclear factor 4 provokes expression of epithelial marker genes, acting as a morphogen in dedifferentiated hepatoma cells. J. Cell Biol. 140:935-946.[Abstract/Free Full Text]





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