Phosphoenolpyruvate Carboxykinase Gene In Liver Research Articles

Vitamin A Status in Mice Affects the Histone Code of the Phosphoenolpyruvate Carboxykinase Gene in Liver

Vitamin A deficiency decreases hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression in mice, and expression is restored with retinoic acid (RA) treatment in vivo. In the studies reported here, we examined changes in histone modification and coregulator association with the regulatory domains of the PEPCK gene in response to alterations in vitamin A status. We identified nuclear receptors that bind to retinoic acid response elements (RAREs) in the PEPCK promoter by electrophoretic mobility shift assay and verified these in vivo using chromatin immunoprecipitation in mouse liver. Hypothetically, nuclear receptors at PEPCK RAREs recruit specific coactivator molecules that contribute to the acetylation of core histones and/or serve as bridging molecules between nuclear receptors and basal transcription factors at the transcription start site. We identified 3 coactivator molecules, cAMP-response element binding protein (CBP), steroid receptor coactivator (SRC)-1, and peroxisome-proliferator activated receptor (PPAR)-gamma-coactivator (PGC)-1alpha, that bound in association with the PEPCK RAREs in vivo. Furthermore, there was differential binding of these coactivators in vitamin A-deficient mice. Related to this, specific lysine residues were acetylated on histones H3 and H4 at the 3 RAREs of the PEPCK promoter, consistent with the action of the above coactivators, and acetylation of certain lysines was significantly decreased with vitamin A deficiency. These results demonstrate the associated changes that occur in nuclear receptor binding, coactivator recruitment, and histone acetylation in response to vitamin A status, identified at specific RAREs in the PEPCK gene in vivo.

Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all- trans or 9- cis retinoic acid (RA) treatment. In a transgenic mouse model, a −460 and −355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all- trans RA treatment. However, there is a differential effect of 9- cis RA on the PEPCK promoter; the −460 fragment confers responsiveness to 9- cis RA, but the −355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9- cis RA induction—but not all- trans RA induction—of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the −355 bp PEPCK promoter fragment did not respond to 9- cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)α and β and the retinoid X receptor (RXR)α—the major retinoid receptors in liver—in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RARβ, but not RARα or RXRα mRNA levels. In situ hybridization showed that RARα, RARβ and RXRα mRNAs were localized in the periportal region, however, immunohistochemistry showed that RARα and RXRα were distributed evenly across the liver acinus, whereas only RARβ levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.

Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by troglitazone: a peroxisome proliferator-activated receptor-γ (PPARγ)-independent, antioxidant-related mechanism

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis. Enhanced expression of the PEPCK gene in liver is present in most models of diabetes, and is thought to contribute to the increased hepatic glucose output seen in this disease. Recently, we showed that troglitazone, the first thiazolidinedione (TZD) used clinically, inhibits expression of the PEPCK gene in isolated hepatocytes. We have pursued the molecular mechanism whereby troglitazone exerts this inhibition. TZDs are known to bind and activate peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor, which regulates expression of target genes. Initially, we examined the abilities of three other TZDs (rosiglitazone, englitazone, and ciglitazone) to inhibit expression of the PEPCK gene. Despite the fact that these agents are ligands for PPARγ, they displayed little if any inhibitory activity on the expression of this gene. GW1929 [ N-(2-benzoyl phenyl)-l-tyrosine], another potent PPARγ ligand that is unrelated structurally to TZDs, had no inhibitory effect on PEPCK gene expression, while a natural PPARγ ligand, the prostaglandin metabolite 15-PGJ 2 (15-deoxy-Δ 12,14-prostaglandin J 2), displayed only modest inhibitory activity. Treatment of hepatocytes with ligands for other isoforms of PPAR also had no significant effect on PEPCK gene expression. Troglitazone has an α-tocopherol (vitamin E) moiety that is not present in other TZDs, and treatment of hepatocytes with vitamin E led to an inhibition of PEPCK gene expression. These observations support the conclusion that troglitazone inhibits the expression of the PEPCK gene by a PPARγ-independent, antioxidant-related mechanism.

Postnatal Profiles of Glycogenolysis and Gluconeogenesis Are Modified in Rat Pups by Maternal Dietary Glucose Restriction

Because glucose is an important metabolic fuel during perinatal development, the effect of restriction of maternal dietary glucose on the developmental profile of neonatal glucoregulatory pathways was investigated. Pregnant rats were fed isoenergetic diets (0, 12, 24 or 60% glucose) and offspring were killed at seven postpartum time periods: 0–2, 4–6, 12–16 and 24 h, and 3, 6 and 15 d. Failure of the most restricted pups (0%) to survive 24 h was explained by persistent hypoglycemia resulting from the following: 1) insufficient tissue glycogen reserves at birth; 2) lower liver glycogen mobilization; 3) delayed phosphorylase a induction; and 4) low phosphoenolpyruvate carboxykinase (PEPCK) gene expression, all of which occurred despite the lower insulin:glucagon ratio. Differences in liver glycogen stores, which had been exhausted in all dietary groups by 16 h, could not account for the high d 1 pup mortality in the moderately restricted (12 and 24% glucose) groups. However, a certain metabolic distress was suggested because these moderately restricted neonates had significantly higher liver PEPCK gene expression at 12–16 h but significantly lower plasma glucose at 24 h. The high d 3 mortality, confirmed by analysis of deviance, was not supported by significant differences in any of the measured glucoregulatory indices. We conclude that dietary glucose during pregnancy is required for neonatal survival; its restriction not only lowers tissue glycogen reserves, but can disrupt the normal gene expression of liver PEPCK and the neonatal profile of phosphorylase a activity. Importantly, these observations show that the development of neonatal glucoregulatory mechanisms is modified by the availability of maternal dietary glucose.

Developmentally specific effects of the DNA cross-linking agent mitomycin C on phosphoenolpyruvate carboxykinase gene expression in vivo: correlation with changes in chromatin structure within the promoter region of the gene.

Previous studies in our laboratory have demonstrated that genotoxic chemical carcinogens have strong preferential effects on expression of certain inducible genes at nonovertly toxic doses in vivo. The effects of the DNA cross-linking agent and chemotherapy drug, mitomycin C (MMC), on expression of the developmental and hormone-regulated gene, phosphoenolpyruvate carboxykinase (PEPCK), were examined in chick embryo liver in vivo as a function of development and were compared with changes in the chromatin structure of the PEPCK gene promoter. The liver PEPCK gene was fully hormone inducible as early as 8 days of embryonic development but was refractory to MMC until after day 10. This onset of responsiveness to MMC was correlated with qualitative changes in the pattern of DNase I hypersensitive sites (DHS) within the PEPCK promoter. There was also a gradual decrease and then a complete loss of both hormone inducibility and MMC responsiveness between 14 and 17 days of development that was correlated with a quantitative change in the overall DNase sensitivity of the liver PEPCK gene promoter over this period. These results suggest that carcinogen sensitivity of the PEPCK gene is related to its ability to respond to its normal induction signals and that chromatin structure may play a central role in these effects.

The Phosphorylation State of the cAMP Response Element Binding Protein Is Decreased in Diabetic Rat Liver

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis. This metabolically important enzyme is unique in that it has no known allosteric modifiers, and all of the regulation of its activity is exerted at the level of gene expression. The expression of the PEPCK gene in liver is elevated in most forms of diabetes, and plays a major contributory role in the hyperglycemia characteristic of this disease. In this study, we initiated studies to determine the molecular basis for the increased PEPCK gene expression in diabetes. RNase protection assays of RNA isolated from control, streptozotocin-induced diabetic, and insulin-treated diabetic rat liver indicated that PEPCK mRNA levels are elevated two- to threefold in diabetic rat liver compared to controls. Nuclear run-on assays indicated that the increased PEPCK mRNA levels can be fully accounted for by changes in the transcription rate of the gene. We next initiated characterization of the cAMP response element binding protein (CREB) in diabetic rat liver, since it is known to play a major role in mediating the basal transcriptional activity of the PEPCK gene as well as the cAMP-dependent stimulation of PEPCK gene transcription, the latter through the phosphorylation of serine 133 of CREB. Western blot analysis of nuclear lysates prepared from rat livers indicated that CREB protein levels in diabetic rat liver nuclei were similar to those of controls. However, using an antibody which specifically recognizes the serine 133-phosphorylated form of CREB, we found that the levels of phospho-CREB were significantly decreased in diabetic rat liver, an effect which insulin treatment reversed. This observation suggests that overexpression of the PEPCK gene in diabetes is not linked to the cAMP signaling system in liver.

Predominant periportal expression of the phosphoenolpyruvate carboxykinase gene in liver of fed and fasted mice, hamsters and rats studied by in situ hybridization

Zonal expression of phosphoenolpyruvate carboxykinase (PCK) mRNA in mouse, hamster and rat liver was studied by in situ hybridization with a radiolabelled rat antisense RNA probe. The abundance of PCK mRNA was determined by Northern blot analysis of total RNA with a digoxigenin-labelled probe. Livers were taken from animals that were sacrificed during the normal day/night cycle and after 29 h fasting. In situ hybridization revealed a heterogeneous distribution pattern of PCK mRNA in the liver of all three species throughout the whole day/night cycle. At the end of the dark period, i.e. at the end of feeding, with rats and mice but at a point of continuous feeding with hamsters, low amounts of PCK mRNA were restricted mainly to the periportal area. At the end of the light period, i.e. at the end of fasting with rats and mice but at a point of continuous feeding with hamsters, PCK mRNA levels were increased to a maximum and extended from the periportal to the intermediate zone. In mouse liver prolonged fasting caused a significant increase in PCK mRNA abundance with a nearly homogeneous distribution within the parenchyma. In hamster and rat liver, however, PCK mRNA levels slightly declined or remained constant, respectively, and the predominant localization of PCK mRNA in the periportal and intermediate zone was preserved. The present data suggest that the heterogeneous zonal activation of the PCK gene was essentially very similar in mouse, hamster and rat liver.

Metabolic Adaptations to Change of Nutrition at Birth

Birth represents a dramatic change of nutrition from a fetal diet rich in carbohydrates and poor in fat to a neonatal diet rich in fat and poor in carbohydrates. Gluconeogenesis and ketogenesis are absent or very low in the fetal liver when the mother is correctly fed, and these metabolic pathways emerge after birth to reach adult values after 24 h. Gluconeogenesis increases rapidly in the liver of the newborn in parallel with the appearance of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of this metabolic pathway. The rise in plasma glucagon, the fall in plasma insulin and the resulting increase in liver cAMP which occur immediately after birth are the factors which induce the activation of liver PEPCK gene transcription. The appearance of ketogenesis is also controlled by the changes of plasma insulin and glucagon that increase the capacity for liver fatty acid oxidation by decreasing lipogenesis and malonyl-CoA concentration, by reducing the sensitivity of carnitine palmitoyl-CoA I to the inhibitory influence of malonyl-CoA, and by activating hydroxymethylglutaryl-CoA synthase by desuccinylation. Once liver PEPCK has reached adult value, i.e. 12 h after birth, other factors are involved in the regulation of hepatic gluconeogenesis. Indeed, the supply of gluconeogenic substrates and of free fatty acid is of crucial importance to support a high rate of gluconeogenesis and to maintain normoglycemia in the newborn. In the liver, fatty acid oxidation provides essential co-factors (acetyl-CoA, NADH and ATP) to support gluconeogenesis, and in peripheral tissue fatty acid oxidation inhibits glucose oxidation and stimulates the production of gluconeogenic precursors (lactate, pyruvate and alanine). Similar mechanisms are operative in human newborn. A defective hepatic fatty acid oxidation is likely to explain the frequent hypoglycemia observed in small-for-date neonates. Administration of oral triglycerides is an efficient mean to prevent hypoglycemia in these newborns.