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The Journal of Nutrition Vol. 127 No. 7 July 1997, pp. 1274-1278
Copyright ©1997 by the American Society for Nutritional Sciences

Vitamin A Regulates Genes Involved in Hepatic Gluconeogenesis in Mice: Phosphoenolpyruvate Carboxykinase, Fructose-1,6-bisphosphatase and 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase1,2

Dong-Ju Shin and Mary M. McGrane3

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

We examined the effects of vitamin A deficiency and all-trans retinoic acid (RA) supplementation on regulation of three important genes in hepatic gluconeogenesis: the genes for phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (Fru-1,6-P2ase) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2ase). Mice were made vitamin A deficient in the second generation by initiating a vitamin A-deficient diet on d 10 of gestation. At 7 wk of age, vitamin A-deficient mice were treated with all-trans RA or vehicle alone and killed for RNA analysis. In liver, vitamin A deficiency resulted in PEPCK mRNA levels that were 74% lower and 6-PF-2-K/Fru-2,6-P2ase mRNA levels that were 42% lower than the respective mRNA measured in control mice. The Fru-1,6-P2ase mRNA abundance was not affected by vitamin A deficiency. The decrease in hepatic PEPCK and 6-PF-2-K/Fru-2,6-P2ase mRNA levels was reversed by treatment with all-trans RA within 3 h of administration. In mice fed the control diet, food deprivation for 15 h resulted in PEPCK mRNA levels that were 3.5-fold higher, Fru-1,6-P2ase mRNA levels that were 2-fold higher, and 6-PF-2-K/Fru-2,6-P2ase mRNA levels that were 3.4-fold higher than in fed mice. Vitamin A-deficient mice did not respond to food deprivation with induced PEPCK mRNA levels, whereas 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase mRNA levels were induced. The pattern of 6-PF-2-K/Fru-2,6-P2ase mRNA abundance with vitamin A deficiency and food deprivation was complex and different from that for either PEPCK or Fru-1,6-P2ase transcripts. The cAMP-responsiveness of the PEPCK gene in vitamin A-deficient mice was tested. Vitamin A deficiency caused a significant reduction in cAMP stimulation of PEPCK mRNA levels in liver. These results in the whole animal indicate that vitamin A regulation of the hepatic PEPCK gene is physiologically important; without adequate vitamin A nutriture, stimulation of the PEPCK gene by food deprivation or cAMP treatment is inhibited in the liver.

KEY WORDS: vitamin A · phosphoenolpyruvate carboxykinase gene · fructose-1,6-bisphosphatase gene · 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene · mice

INTRODUCTION

Retinoids, compounds that bind to a specific set of receptors and have physiological activity ascribed to vitamin A, function as signaling molecules in vertebrate development and differentiation (Thaller and Eichele 1990, Tickle et al. 1982). Recently, it has been shown that retinoids regulate the gene for a rate-determining enzyme in hepatic gluconeogenesis, cytosolic phosphoenolpyruvate carboxykinase (GTP) (PEPCK,4 EC 4.1.1.32) from rats (Hall et al. 1992, Lucas et al. 1991a and 1991b, Pan et al. 1990, Shin et al. 1995). The naturally occurring retinoids in the nucleus, all-trans retinoic acid (RA) and the stereoisomer, 9-cis RA, activate their respective receptors, the RA receptors (RAR) and retinoid X receptors (RXR), which function as nuclear transcription factors (Heyman et al. 1992, Leid et al. 1992a and 1992b). These receptors are members of the steroid/thyroid/retinoid superfamily of nuclear receptors that bind as dimers to specific hormone response elements (HRE) in target genes (Giguere et al. 1987, Mangelsdorf et al. 1991, Perlman et al. 1993). One of these target genes, the gene that encodes PEPCK, contains specific HRE in its 5' flanking sequence and is tissue-specifically regulated by retinoids in liver. Retinoid regulation of the PEPCK gene may be involved in the developmental onset of PEPCK gene transcription in late gestation, as well as the maintenance of basal tissue-specific expression after birth. The well-known connection between vitamin A deficiency and hepatic glycogen depletion caused by reduced gluconeogenesis can now be explained at the molecular level by the dependence of PEPCK gene expression on adequate vitamin A.

All-trans RA and 9-cis RA increase PEPCK gene transcription in H4IIE rat hepatoma cells, and two RA response elements (RARE) within the PEPCK 5' flanking sequence have been described (Hall et al. 1992, Lucas et al. 1991a and 1991b, Scott et al. 1996). Recently, we demonstrated the effects of vitamin A deficiency and all-trans RA supplementation on expression of a chimeric PEPCK/bovine growth hormone (bGH) gene in transgenic mice (Shin et al. 1995). The later studies showed that retinoid regulation of a defined region of the PEPCK promoter occurs in vivo. It is possible, therefore, that other liver-specific genes that encode enzymes involved in gluconeogenesis may be regulated by vitamin A. In this study we report the effects of vitamin A deficiency and all-trans RA supplementation on mRNA levels for three genes that encode gluconeogenic enzymes: the PEPCK gene, the fructose-1,6-bisphosphatase (Fru-1,6-P2ase) gene, and the liver-specific (L) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2ase) gene.

MATERIALS AND METHODS

Materials. Dibutyryl adenosine 3'-5'-cyclic monophosphate (Bt2cAMP) was from Boehringer Mannheim (Indianapolis, IN), and theophylline was from Calbiochem (La Jolla, CA). [alpha -32P]dCTP (29.6 GBq/µmol) was purchased from Du Pont NEN (Boston, MA), as were the Gene Screen Plus membranes used for Northern blots.

Animals and diets. C57BL/6 × SJL mice (Jackson Laboratory, Bar Harbor, ME) were used. Mice were randomly selected for vitamin A-deficient and control groups: vitamin A-deficient mice were fed an AIN-76A vitamin A-deficient diet (Lamb et al. 1974), and control mice were fed the AIN-76A diet with 3600 retinol equivalents of retinyl esters per kilogram of diet (Dyets Inc., Bethlehem, PA). The mice were made vitamin A deficient by feeding pregnant females the chemically defined AIN-76A diet lacking vitamin A from d 10 of gestation through the lactation period. When mice were 7 wk old, vitamin A deficiency was verified by determining retinol concentrations in the blood by HPLC (Furr et al. 1986). Serum retinol concentrations ranged from 105 to 222 nmol/L, indicative of vitamin A deficiency. Control mice had serum retinol concentrations that ranged from 734 to 816 nmol/L. Mice were given free access to the pelleted diets unless food deprivation studies were conducted. Mice were maintained at 24°C on a 12:12 h dark-light schedule. The care of the animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee of the University of Connecticut.

At 7 wk of age, vitamin A-deficient mice were treated by gavage with all-trans RA (Sigma Chemical, St. Louis, MO) (50 mg/kg body wt) in peanut oil or with vehicle alone. At this time mice had average weights of 20.1 ± 1.9 and 22.5 ± 2.9 g in the control and vitamin A-deficient groups, respectively. At 3, 6 and 12 h after treatment, mice were killed by cervical dislocation. The livers were removed and immediately placed in liquid nitrogen for subsequent RNA extraction. Vitamin A-deficient mice, vitamin A-deficient mice with all-trans RA treatment, and control mice were subjected to either 12 h of food deprivation or a time course of food deprivation for 6, 9 and 15 h before they were killed for RNA extraction.

RNA analysis. Total RNA was isolated from mouse livers by the acid guanidinium-phenol extraction method of Chomszynski and Sacchi (1987). For Northern blot analysis, 10 or 20 µg of total RNA was loaded onto an agarose gel (10 g/L, 0.66 mol/L formaldehyde) for electrophoresis. A Posiblot apparatus (Stratagene, La Jolla, CA) was used to transfer RNA to Gene Screen Plus (DuPont NEN) membranes. After UV crosslinking, the membranes were hybridized with 32P-labeled PEPCK, Fru-1,6-P2ase, and 6-PF-2-K/Fru-2,6-P2ase cDNA probes. A 1.6-kb fragment of the ribosomal protein, rpl32, cDNA was used as probe to assay total RNA per lane. After hybridization, the filters were exposed to Kodak XAR-5 film at -80°C. Autoradiograms were quantified by scanning with a BIO RAD model GS-670 imaging densitometer; the resulting images were analyzed using Molecular Analyst software (BIO RAD, Melville, NY).

The following cDNA probes were used: 3'-PEPCK, a 1.1-kb Pst I fragment from the 3' end of the rat PEPCK cDNA; Fru-1,6-P2ase, a 1.2-kb Xba I fragment from the rat cDNA (the generous gift of R. El-Maghrabi, SUNY, Stoney Brook, NY); and 6-PF-2-K/Fru-2,6-P2ase, an 1.6-kb Nde I-Hind III fragment from the rat L-type cDNA (the generous gift of S. Pilkis, University of Minnesota, MN).

cAMP Treatment. Vitamin A-deficient and control mice were injected intraperitoneally with 30 mg/kg body wt of Bt2cAMP and 30 mg/kg theophylline at three consecutive 30-min intervals to test the response to maximal cAMP stimulation (Lamers et al. 1982). After 90 min, the mice were killed for RNA extraction from the liver. The PEPCK mRNA levels were measured after cAMP treatment, and comparisons were made between vitamin A-deficient and control mice.

Statistical analysis. Results are reported as means ± SEM. Differences between means were determined using the Student's t test.

RESULTS

Hepatic PEPCK, 6-PF-2-K/Fru-2,6-P2ase, and Fru-1,6-P2ase mRNA levels in mice with vitamin A deficiency and all-trans RA supplementation. Hepatic PEPCK, 6-PF-2-K/Fru-2,6-P2ase, and Fru-1,6-P2ase mRNA levels were measured in control and vitamin A-deficient mice after 12 h of food deprivation. Under these conditions, PEPCK mRNA levels were 74% lower in vitamin A-deficient mice than in mice fed the control diet (P < 0.005) (Fig. 1). L-type 6-PF-2-K/Fru-2,6-P2ase mRNA levels were 42% lower in vitamin A-deficient mice than in mice fed the control diet (P < 0.005) (Fig. 1). However, Fru-1,6-P2ase mRNA levels were not affected by vitamin A deficiency (Fig. 1).
Fig. 1. The effect of vitamin A deficiency in mice on hepatic mRNA levels for the phosphoenolpyruvate carboxykinase (PEPCK), the fructose-1,6-bisphosphatase (Fru-1,6-P2ase) and the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2ase) genes. Total RNA was isolated from livers of mice fed either the control or vitamin A-deficient diet, and Northern blot analysis was performed as described in Materials and Methods. The mRNA levels were determined by densitometric scan of representative autoradiograms. Any difference in the amount of total RNA per lane was corrected for by ratio to mRNA levels of the ribosomal protein, rpl32. PEPCK, Fru-1,6-P2ase and 6-PF-2-K/Fru-2,6-P2ase mRNA levels from mice fed the control diet were set at arbitrary values of 1.0, and the relative change in respective mRNA abundance in livers of vitamin A-deficient mice is indicated. Values are means ± SEM, n = 6. *Significantly different from controls (P < 0.005).
[View Larger Version of this Image (43K GIF file)]

To determine whether the observed changes in hepatic mRNA levels with vitamin A deficiency were reversed by all-trans RA, vitamin A-deficient mice were treated with all-trans RA and specific mRNA levels measured at 3, 6, 12 and 24 h after treatment. Hepatic PEPCK mRNA levels were 3.4-fold higher at 3 h, 2.2-fold higher at 6 h, and 3.6-fold higher at 12 h, relative to PEPCK mRNA levels in untreated vitamin A-deficient mice killed at the same time (P < 0.001) (Fig. 2). Treatment with all-trans RA returned hepatic PEPCK mRNA levels to equal to (or greater than) those measured in mice fed the control diet, and maximal stimulation occurred by 3 h. However, by 24 h, PEPCK mRNA levels were decreased to those found in untreated, vitamin A-deficient mice (data not shown). L-type 6-PF-2-K/Fru-2,6-P2ase mRNA levels were 90% higher at 3 h after all-trans RA treatment compared with 6-PF-2-K/Fru-2,6-P2ase mRNA levels in untreated vitamin A-deficient mice (P < 0.05) (Fig. 2). However, at 6 and 12 h, all-trans RA treatment did not change 6-PF-2-K/Fru-2,6-P2ase mRNA levels compared with those of untreated vitamin A-deficient mice. Fru-1,6-P2ase mRNA levels were not significantly affected by all-trans RA treatment (Fig. 2).

Fig. 2. Hepatic phosphoenolpyruvate carboxykinase (PEPCK), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2ase) and fructose-1,6-bisphosphatase (Fru-1,6-P2ase) mRNA levels following treatment of vitamin A-deficient mice with all-trans retinoic acid (RA) by gavage. Mice from both groups were killed at 3, 6, and 12 h after treatment. Total RNA was isolated from livers and Northern blot analysis was performed as described in Materials and Methods. The mRNA levels were determined by densitometric scan of representative autoradiograms. Any difference in the amount of total RNA per lane was corrected for by ratio to mRNA levels of the ribosomal protein, rpl32. PEPCK, 6-PF-2-K/Fru-2,6-P2ase and Fru-1,6-P2ase mRNA levels from mice fed the vitamin A-deficient diet were set at arbitrary values of 1.0, and the relative change in respective mRNA abundance in livers of all-trans RA-treated mice is indicated. Values are means ± SEM, n = 6. *Significantly different from A- mice (P < 0.001, PEPCK; P < 0.05, 6-PF-2-K/Fru-2,6-P2ase).
[View Larger Version of this Image (20K GIF file)]

Effect of food deprivation on hepatic PEPCK and 6-PF-2-K/Fru-2,6-P2ase mRNA levels with vitamin A deficiency and all-trans RA supplementation. Hepatic PEPCK mRNA levels were induced with food deprivation over a 15-h time course in control mice. At 6 h of food deprivation, hepatic PEPCK mRNA levels in control mice and mice fed the vitamin A-deficient diet were not significantly different (Fig. 3) and resembled levels in fed mice (data not shown). At 9 h of food deprivation, hepatic PEPCK mRNA levels in control mice were increased 2.1-fold over 6-h levels; vitamin A-deficient mice exhibited a 1.7-fold increase. After 15 h of food deprivation, PEPCK mRNA levels in control mice were elevated 3.5-fold over 6-h levels. In contrast, PEPCK mRNA levels in vitamin A-deficient mice were not different from those measured at 6 h (Fig. 3). Treatment of vitamin A-deficient mice with all-trans RA, however, elevated PEPCK mRNA levels at 6, 9 and 15 h of food deprivation by 3.5-fold, 3.9-fold, and 3.4-fold respectively, over levels measured at 6 h in untreated mice (P < 0.001) (Fig. 3).
Fig. 3. The effect of food deprivation on hepatic phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels in control mice, vitamin A-deficient mice, and vitamin A-deficient mice treated with all-trans retinoic acid (RA). Upper panel: Representative autoradiogram of a Northern blot of total RNA (10 µg/lane) isolated from livers of mice fed the control diet (A+), the vitamin A-deficient diet (A-) and the vitamin A-deficient diet + all-trans RA treatment (A-/+RA). Mice from the above groups were killed at 6, 9 and 15 h of food deprivation (as indicated). The Northern blot was hybridized with a 32P-labeled PEPCK cDNA probe. The PEPCK mRNA of 2.8 kb is indicated. Any difference in the amount of total RNA per lane was corrected for by ratio to mRNA levels of the ribosomal protein, rpl32. Lower panel: Quantification of PEPCK mRNA levels by densitometric scan. Mean values for PEPCK mRNA levels are given for control mice (black bars), vitamin A-deficient mice (striped bars) and vitamin A-deficient mice treated with all-trans RA (gray bars). Values are means ± SEM, n = 6.
[View Larger Version of this Image (49K GIF file)]

L-type 6-PF-2-K/Fru-2,6-P2ase mRNA levels also increased with food deprivation in control mice. At 15 h of food deprivation, 6-PF-2-K/Fru-2,6-P2ase mRNA levels were 3.4-fold greater than 6 h levels (Fig. 4). In mice fed the vitamin A-deficient diet, at 6 and 9 h of food deprivation, 6-PF-2-K/Fru-2,6-P2ase mRNA levels did not differ from 6-PF-2-K/Fru-2,6-P2ase mRNA levels in mice fed the control diet (Fig. 4). However, by 15 h of food deprivation, 6-PF-2-K/Fru-2,6-P2ase mRNA levels in vitamin A-deficient mice were increased only 1.6-fold over 6-h levels (3.4-fold in controls vs. 1.6-fold in deficient mice, P < 0.005) (Fig. 4). Additionally, with 6 h of food deprivation, all-trans RA treatment of vitamin A-deficient mice caused 6-PF-2-K/Fru-2,6-P2ase mRNA levels to increase 2.5-fold (P < 0.005), but at 15 h of food deprivation, treatment of vitamin A-deficient mice with all-trans RA did not increase 6-PF-2-K/Fru-2,6-P2ase mRNA levels significantly (Fig. 4).

Fig. 4. The effect of food deprivation on hepatic 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6-PF-2-K/Fru-2,6-P2ase) mRNA levels in control mice (A+), vitamin A-deficient mice (A-), and vitamin A-deficient mice treated with all-trans retinoic acid (RA) (A-/+RA). Quantification of 6-PF-2-K/Fru-2,6-P2ase mRNA levels was by densitometric scan. Values are means ± SEM, n = 6.
[View Larger Version of this Image (48K GIF file)]

Vitamin A deficiency and cAMP-responsiveness of the PEPCK gene in liver. Because hepatic PEPCK mRNA levels were not induced by food deprivation in mice with vitamin A deficiency, we tested the cAMP-responsiveness of the PEPCK gene in vitamin A-deficient mice. Vitamin A-deficient mice and mice fed the control diet were intraperitoneally injected with Bt2cAMP (Materials and Methods). As shown in Figure 5, PEPCK mRNA levels in livers from Bt2cAMP-treated mice fed the control diet were elevated 3.7-fold over those measured in untreated mice. However, only a 2.5-fold increase in PEPCK mRNA levels was measured in vitamin A-deficient mice after Bt2cAMP treatment. The difference in Bt2cAMP stimulation of PEPCK mRNA levels between vitamin A-deficient mice and control mice was significant (P < 0.005).
Fig. 5. Comparison of cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene in control (A+) and vitamin A deficient mice (A-). Upper panel: Representative autoradiogram of a Northern blot of total RNA (10 µg/lane) isolated from livers of mice fed the control diet (A+) or the vitamin A-deficient diet (A-) treated with or without dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) (as indicated). The Northern blot was hybridized with a 32P-labeled PEPCK cDNA probe. The PEPCK mRNA of 2.8 kb is indicated. Any difference in the amount of total RNA per lane was corrected for by ratio to mRNA levels of the ribosomal protein, rpl32. Lower panel: Quantification of PEPCK mRNA levels by densitometric scan. Mean values for PEPCK mRNA levels are given for untreated control (A+) and vitamin A-deficient (A-) mice (black bars) and control and vitamin A-deficient mice treated with Bt2cAMP (stripped bars). Values are means ± SEM, n = 6.
[View Larger Version of this Image (45K GIF file)]

DISCUSSION

Our results indicate that PEPCK gene expression is inhibited by vitamin A deficiency and stimulated by all-trans RA supplementation, whereas Fru-1,6-P2ase gene expression is not inhibited by vitamin A deficiency. The 6-PF-2-K/Fru-2,6-P2ase gene is responsive to vitamin A status, albeit the pattern is less clear than for the PEPCK gene. This is the first report to show that the endogenous gene for a rate-determining enzyme in hepatic gluconeogenesis (the PEPCK gene) is regulated by vitamin A and is specifically induced by all-trans RA treatment in the whole animal. It is well documented that PEPCK enzyme activity is directly related to the amount of PEPCK protein, and the amount of PEPCK protein is determined primarily by the rate of transcription of the PEPCK gene and the amount of PEPCK mRNA (Granner et al. 1991). Therefore, retinoid regulation of PEPCK mRNA levels is predicted to regulate PEPCK enzyme activity with subsequent effects on liver glucose metabolism. Our results are consistent with a much earlier study that showed hepatic gluconeogenesis from [14C]lactate is impaired in vitamin A-deficient rats (Wolf et al. 1957).

The second gluconeogenic gene tested, that for Fru-1,6-P2ase, is not inhibited by vitamin A deficiency. Although both PEPCK and Fru-1,6-P2ase catalyze irreversible reactions in the gluconeogenic pathway in liver, the genes that encode these enzymes have a different developmental expression profile. There is Fru-1,6-P2ase activity, but not PEPCK activity, in the fetal liver. The lack of PEPCK activity during fetal development is due to inhibition of transcription of the PEPCK gene by the high insulin/glucagon ratio (Garcia-Ruiz et al. 1978). At birth, the insulin/glucagon ratio declines, the PEPCK gene is activated, and the gluconeogenic pathway in liver begins to function. It has been reported that during late gestation in rats (d 18), the first segment of the PEPCK promoter to bind a nuclear protein from liver is the segment between -456 and -433 bp from the start site of transcription (Trus et al. 1990). The later DNA sequence corresponds to the RARE determined in transfected H4IIE hepatoma cells between -451 and -433 bp (Lucas et al. 1991b). Hypothetically, all-trans RA, by activating nuclear retinoid receptors that bind to the PEPCK promoter, initiates the transition to competence of the PEPCK gene during liver cytodifferentiation. In the competent state, the PEPCK gene then has the capacity to respond to later hormonal stimuli, such as the change in insulin/glucagon ratio that occurs at birth. In the adult liver, retinoids may be required to maintain basal liver-specific expression of the PEPCK gene.

The third gene investigated, that for L-type 6-PF-2-K/Fru-2,6-P2ase, codes for a bifunctional enzyme that catalyzes both the synthesis and breakdown of Fru-2,6-P2 , a key allosteric regulator of hepatic glycolysis and gluconeogenesis. Fru-2,6-P2 inhibits Fru-1,6-P2ase activity and stimulates 6-phosphofructo-1-kinase (6-PF-1-K) activity. Because 6-PF-2-K/Fru-2,6-P2ase is bifunctional, it either increases glycolysis or gluconeogenesis depending upon which activity is predominant. In the short-term, 6-PF-2-K/Fru-2,6-P2ase activity is regulated by cAMP-dependent phosphorylation, which activates the Fru-2,6-P2ase activity. In the long-term, 6-PF-2-K/Fru-2,6-P2ase activity is regulated by changes in 6-PF-2-K/Fru-2,6-P2ase gene expression. It has been reported that in rat liver, the amount of 6-PF-2-K/Fru-2,6-P2ase protein is decreased with 24 h of starvation, although there is no change in 6-PF-2-K/Fru-2,6-P2ase mRNA levels (Colosia et al. 1988). However, our results with short-term food deprivation in mice indicate that hepatic 6-PF-2-K/Fru-2,6-P2ase mRNA levels increase during 15 h of food deprivation. The increase in 6-PF-2-K/Fru-2,6-P2ase mRNA abundance in liver is similar to that for the two gluconeogenic genes measured, those encoding PEPCK and Fru-1,6-P2ase (data not shown). The vitamin A status of the mouse, however, does not have the same effect on the 6-PF-2-K/Fru-2,6-P2ase gene as on either the PEPCK or Fru-1,6-P2ase genes. Vitamin A deficiency seems to decrease 6-PF-2-K/Fru-2,6-P2ase mRNA levels only when they are induced after 15 h of food deprivation. On the other hand, at 6 h of food deprivation, when the 6-PF-2-K/Fru-2,6-P2ase mRNA levels are low, all-trans RA treatment of vitamin A-deficient mice increases 6-PF-2-K/Fru-2,6-P2ase mRNA levels by more than twofold over untreated levels. Therefore, although vitamin A deficiency does not inhibit 6-PF-2-K/Fru-2,6-P2ase gene expression in the early stages of food deprivation, all-trans RA treatment is stimulatory. A more detailed investigation of the effects of food deprivation on the 6-PF-2-K/Fru-2,6-P2ase gene in mouse liver, and the effect of vitamin A deficiency thereon, needs to be conducted to determine the overall importance of these findings.

Cumulative evidence makes it clear that the PEPCK gene is regulated by all-trans RA. Retinoid regulation of the PEPCK gene has been measured in transformed hepatocyte cell lines (Pan et al. 1990), in transfected H4IIE hepatoma cells (Hall et al. 1992, Lucas et al. 1991a and 1991b, Scott et al. 1996) and in a PEPCK/bGH transgenic mouse model (Shin et al. 1995). In the present study we report that the endogenous PEPCK gene is inhibited in mice with vitamin A deficiency and stimulated by all-trans RA treatment. Vitamin A deficiency also decreases the cAMP-responsiveness of the PEPCK gene. This result is consistent with the findings of Pan et al. (1990), who reported a positive effect of all-trans RA on cAMP stimulation of PEPCK mRNA levels in an immortalized rat hepatocyte cell line. Granner and colleagues have shown that the PEPCK promoter contains two RARE, one located from -451 to -433 bp (RARE 1) and a second located from -337 to -321 bp (RARE 2) (Scott et al. 1996). The latter RARE is physiologically important, because transgenic mice containing a PEPCK/bGH gene with a PEPCK promoter truncated to -355 exhibit changes in transgene mRNA abundance in response to changes in vitamin A status (Shin et al. 1995). It is possible that binding of activated nuclear retinoid receptors to one or two of the RARE of the PEPCK promoter is required for full cAMP stimulation as well. In mouse liver, we find that RARbeta mRNA levels are significantly decreased with vitamin A deficiency and stimulated with all-trans RA treatment (Shin et al. 1995). Therefore, adequate intranuclear RARbeta levels, as well as all-trans RA activation of RARbeta , may be required for appropriate cAMP stimulation of the PEPCK gene in liver.

FOOTNOTES

1   Supported by National Institutes of Health grant DK49682 (to M.M.M.)
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 should be addressed.
4   Abbreviations used: Bt2cAMP, dibutyryl adenosine 3':5'-cyclic monophosphate; bGH, bovine growth hormone; Fru-1,6-P2ase, fructose-1,6-bisphosphatase; Fru-2,6-P2 , fructose 2,6-bisphosphate; HRE, hormone response element; PEPCK, phosphoenolpyruvate carboxykinase; 6-PF-1-K, 6-phosphofructo-1-kinase; 6-PF-2-K/Fru-2,6-P2ase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor.

Manuscript received 3 July 1996. Initial reviews completed 12 August 1996. Revision accepted 4 March 1997.

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



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Vitamin A Depletion Is Associated with Low Phosphoenolpyruvate Carboxykinase mRNA Levels during Late Fetal Development and at Birth in Mice
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Hepatic Vitamin A Depletion Is a Sensitive Marker of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Exposure in Four Rodent Species
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M. J. Rowling and K. L. Schalinske
Retinoid Compounds Activate and Induce Hepatic Glycine N-Methyltransferase in Rats
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J.-C. Wang, P.-E. Strömstedt, T. Sugiyama, and D. K. Granner
The Phosphoenolpyruvate Carboxykinase Gene Glucocorticoid Response Unit: Identification of the Functional Domains of Accessory Factors HNF3{beta} (Hepatic Nuclear Factor-3{beta}) and HNF4 and the Necessity of Proper Alignment of Their Cognate Binding Sites
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Structural Requirements of the Glucocorticoid and Retinoic Acid Response Units in the Phosphoenolpyruvate Carboxykinase Gene Promoter
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