PEPCK Gene Research Articles

Increased Expression and Activity of the Transcription Factor FOXO1 in Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver, affecting 34% of the U.S. population, is characterized by hepatic insulin resistance, which is more marked in the presence of steatohepatitis, and frequently precedes hyperglycemia. The molecular mechanisms underlying the relationship between fatty liver and insulin resistance are still undergoing definition and have not been evaluated in humans. Our aim was to evaluate the relationship between insulin resistance and the expression and regulation of forkhead box-containing protein O subfamily-1 (FOXO1), a transcription factor that mediates the effect of insulin on the gluconeogenic genes PEPCK and glucose-6-phosphatase catalytic subunit (G6PC). FOXO1, PEPCK, and G6PC mRNA levels were evaluated in 84 subjects: 26 with steatohepatitis, 28 with steatosis alone, 14 with normal liver histology without metabolic alterations, and 16 with hepatitis C virus chronic hepatitis, of whom 8 were with and 8 were without steatosis. Protein expression and regulation of FOXO1 and upstream insulin signaling were analyzed in a subset. RESULTS; Expression of PEPCK was higher in steatohepatitis compared with steatosis alone and normal liver, and it was correlated with the homeostasis model assessment of insulin resistance (HOMA-IR) index. FOXO1 mRNA levels were higher in steatohepatitis, correlated with PEPCK and G6PC mRNA and with HOMA-IR. FOXO1 upregulation was confirmed at protein levels in steatohepatitis and, in the presence of oxidative stress, was associated with decreased Ser(256) phosphorylation, decreased Akt1, and increased Jun NH(2)-terminal kinase-1 activity. Consistently, immunohistochemistry showed increased FOXO1 expression and nuclear localization in steatohepatitis. FOXO1 mRNA levels correlated with nonalcoholic steatohepatitis activity score and were modulated by drugs counteracting hepatic lipogenesis. FOXO1 expression and activity are increased in patients with steatohepatitis, and mRNA levels are correlated with hepatic insulin resistance.

PCK1 and PCK2 as candidate diabetes and obesity genes

The PCK1 gene (Pck1 in rodents) encodes the cytosolic isozyme of phosphoenolpyruvate carboxykinase (PEPCK-C), which is well-known for its function as a gluconeogenic enzyme in the liver and kidney. Mouse studies involving whole body and tissue-specific Pck1 knockouts as well as tissue-specific over-expression of PEPCK-C have resulted in type 2 diabetes as well as several surprising phenotypes including obesity, lipodystrophy, fatty liver, and death. These phenotypes arise from perturbations not only in gluconeogenesis but in two additional metabolic functions of PEPCK-C: (1) cataplerosis which maintains metabolic flux through the Krebs cycle by removing excess oxaloacetate, and (2) glyceroneogenesis which produces glycerol-3-phosphate as a precursor for fatty acid esterification into triglycerides. PEPCK-C catalyzes the conversion of oxaloacetate + GTP to phosphoenolpyruvate + GDP + CO2. It is in part the tissue-specificity of this simple reaction that results in the variety of phenotypes listed above. Briefly: (1) A 7-fold over-expression of PEPCK-C in the livers of mice causes excessive glucose production. (2) Mice with a whole-body knockout of Pck1 die within 2-3 days of birth, not from hypoglycemia, but probably because the Krebs cycle slows to approximately 10% of normal in the absence of cataplerosis. (3) Mice with a liver-specific knockout have an inability to remove oxaloacetate from the Krebs cycle, which leads to a fatty liver following a fast. (4) An adipose-specific knockout of Pck1 results in a fraction of the mice developing lipodystrophy due to lost glyceroneogenesis and a consequent decrease in fatty acid re-esterification. (5) Finally, disregulated over-expression of PEPCK-C in adipose tissue increases fatty acid re-esterification leading to obesity. These varied experimental phenotypes in mice have led us to postulate that abnormal production of PEPCK isozymes encoded by two PEPCK genes, PCK1 and PCK2, in humans could have similar consequences (Beale, E. G. et al. (2004). Trends in Endocrinology and Metabolism, 15, 129-135). The purpose of this review is to further explore these possibilities.

ATF-2 Regulates Fat Metabolism inDrosophila

ATF-2 is a member of the ATF/CREB family of transcription factors that is activated by stress-activated protein kinases such as p38. To analyze the physiological role of Drosophila ATF-2 (dATF-2), we generated dATF-2 knockdown flies using RNA interference. Reduced dATF-2 in the fat body, the fly equivalent of the mammalian liver and adipose tissue, decreased survival under starvation conditions. This was due to smaller triglyceride reserves of dATF-2 knockdown flies than control flies. Among multiple genes that control triglyceride levels, expression of the Drosophila PEPCK (dPEPCK) gene was strikingly reduced in dATF-2 knockdown flies. PEPCK is a key enzyme for both gluconeogenesis and glyceroneogenesis, which is a pathway required for triglyceride synthesis via glycerol-3-phosphate. Although the blood sugar level in dATF-2 knockdown flies was almost same as that in control flies, the activity of glyceroneogenesis was reduced in the fat bodies of dATF-2 knockdown flies. Thus, reduced glyceroneogenesis may at least partly contribute to decreased triglyceride stores in the dATF-2 knockdown flies. Furthermore we showed that dATF-2 positively regulated dPEPCK gene transcription via several CRE half-sites in the PEPCK promoter. Thus, dATF-2 is critical for regulation of fat metabolism.

N-3 Fatty Acids Preserve Insulin Sensitivity In Vivo in a Peroxisome Proliferator–Activated Receptor-α–Dependent Manner

Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet-induced insulin resistance through peroxisome proliferator-activated receptor (PPAR)-alpha activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis, we fed PPAR-alpha null and wild-type mice for 2 weeks with isocaloric high-fat diets containing 27% fat from either safflower oil or safflower oil with an 8% fish oil replacement (fish oil diet). In both genotypes the safflower oil diet blunted insulin-mediated suppression of hepatic glucose production (P < 0.02 vs. genotype control) and PEPCK gene expression. Feeding wild-type mice a fish oil diet restored hepatic insulin sensitivity (hepatic glucose production [HGP], P < 0.002 vs. wild-type mice fed safflower oil), whereas in contrast, in PPAR-alpha null mice failed to counteract hepatic insulin resistance (HGP, P = NS vs. PPAR-alpha null safflower oil-fed mice). In PPAR-alpha null mice fed the fish oil diet, safflower oil plus fish oil, hepatic insulin resistance was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentration (P < 0.0001 vs. genotype control). These data support the hypothesis that n-3 fatty acids protect from high-fat diet-induced hepatic insulin resistance in a PPAR-alpha-and diacylglycerol-dependent manner.

Growth Hormone Corrects Proliferation and Transcription of Phosphoenolpyruvate Carboxykinase in Livers of Old Mice via Elimination of CCAAT/Enhancer-binding Protein α-Brm Complex

Growth hormone (GH), which is reduced with age, corrects the impaired proliferative capacity of livers of old animals. In this paper, we present a mechanism by which GH eliminates age-dependent negative control of proliferation and increases transcription of liver-specific genes in livers of old mice. The reduced proliferative capacities of the liver of old animals are associated with the CCAAT/enhancer-binding protein alpha (C/EBPalpha)-Brm complex, which inhibits E2F-dependent promoters. We found that a sequestration of C/EBPalpha into complexes with Brm leads to a weak interaction of C/EBPalpha with promoters of liver-specific genes, expression of which is reduced in old animals. Injection of either GH or the regulator of the amplitude of endogenous GH release, ghrelin, reduces the C/EBPalpha-Brm complex in livers of old mice, leading to a derepression of E2F targets, to increased interactions of C/EBPalpha with promoters of liver-specific genes, and to correction of their expression. GH-dependent elimination of the complex is mediated by the inhibition of cyclin D3-CDK4 activity and by elevation of a phosphatase, protein phosphatase 2A, which dephosphorylates C/EBPalpha and dissociates the complex.

Nutrient control of phosphorylation and translocation of Foxo1 in C57BL/6 and db/db mice

The nutrient response mediated by feeding or fasting plays an important role in controlling gluconeogenic gene expression such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxylase (PEPCK). The FOXO family of forkhead transcription factor Foxo1 (mouse FOXO1) is a key regulator that stimulates the expression of gluconeogenic genes in the nucleus but is phosphorylated by Akt (also known as protein kinase B; PKB) and translocated to the cytoplasm in response to insulin. Although it has been widely accepted that the cellular signaling of insulin represses Foxo1 function through Akt-dependent phosphorylation, the molecular mechanism behind the modulation of Foxo1 function by nutrient responses, including feeding or fasting, remains unknown in vivo. We investigated the consequences of the nutritional changes in Akt-mediated Foxo1 phosphorylation and translocation in the liver using control C57BL/6 and diabetic db/db mice. We found that feeding promotes the phosphorylation and nuclear exclusion of Foxo1, whereas fasting counteracted them in C57BL/6 mice. Notably, db/db mice exhibited constitutive phosphorylation but dominant nuclear accumulation of Foxo1, even though CREB phosphorylation usually occurred in the fasted status. Furthermore, in contrast to C57BL/6 mice, the expression of G6Pase, PEPCK and PGC-1alpha genes during feeding was not down-regulated in db/db mice. Thus, we suggest that the accurate regulation of Foxo1 via Akt-dependent phosphorylation is required for physiological adaptation to different nutritional statuses.

Establishment of chromatin immunoprecipitation assay to analyze transcriptional regulation of gene expression

目的 建立一种染色质免疫沉淀技术(ChIP)分析肝细胞基因表达的转录调控方法.方法以肝脏特异性基因磷酸烯醇式丙酮酸激酶(PEPCK)为待测基因,以IgG和次黄嘌呤鸟嘌呤磷酸核糖基转移酶(HPRT)及髓磷脂基础蛋白(MBP)作为内参基因,采用ChIP验证不同代谢状态下cAMP应答元件结合蛋白(CREB)对PEPCK基因启动子区的结合情况.结果在抗CREB抗体免疫沉淀的DNA模板中含有一明显增强的DNA片段,即禁食状态下PEPCK基因启动子区域CREB的结合增加4倍,证明DNA片段中含有与PEPCK基因启动子区结合的CREB序列.结论研究证实,CREB蛋白在体内可结合于PEPCK基因的启动子区域,并参与该基因表达的转录调控。

Hormonal regulation of assembly of the pepck gene transcription complex

Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription is activated in response to glucocorticoids, such as dexamethasone (Dex). Previous studies, using a variety of techniques, including reporter gene assays, showed that the induction of PEPCK gene transcription by Dex requires a complex array of DNA accessory elements and associated DNA-binding transcription factors. These, in turn, bind a set of transcription coactivators, including CREB binding protein (CBP) and p300. We have now used the chromatin immunoprecipitation assay (ChIP) to assess promoter occupancy of these proteins, in vivo, in the presence and absence of the ligand, Dex. Dex causes a remarkable increase in the binding of glucocorticoid receptor, polymerase II, CBP and p300 to the promoter. However, even though both CBP and p300 are expressed in the nucleus of H4IIE cells, they do not co-exist in the same transcription complex on the PEPCK gene promoter. Furthermore, the selective reduction of either p300 or CBP, using small interfering RNAs (siRNAs), partially abolishes the Dex response of the PEPCK gene. Reduction of both CBP and p300 totally abolishes the Dex response of the PEPCK gene. Although endogenous p300 and CBP are not capable of compensating for the loss of CBP and p300, respectively, the overexpression of either of these proteins compensates for the loss of the other, and both coactivators support the dominant suppression of the gene by insulin.

Estrogen‐Related Receptor α (ERRα) represses PGC‐1α‐activated PEPCK gene transcription

The orphan nuclear receptor ERRα acts as a downstream effector of the transcriptional coactivator PGC-1α in the induction of genes important for mitochondrial oxidative metabolism. PGC-1α is also known as a potent activator of the transcriptional program required for hepatic gluconeogenesis, and in particular of the key gluconeogenic enzyme PEPCK. We report here that ERRα binding to the PEPCK gene promoter leads to the repression of PEPCK expression. Gain- and loss-of function studies suggest that ERRα at the PEPCK promoter antagonizes the recruitment and stimulatory action of PGC-1α. Consistent with these findings, animals that lack ERRα show increased expression of gluconeogenic genes, including PEPCK and glycerol kinase, but decreased expression of mitochondrial genes. We conclude that ERRα has opposing effects on genes important for mitochondrial oxidative capacity and for gluconeogenesis. Decreased mitochondrial oxidative function and increased gluconeogenesis are associated with diabetes. The different functions of ERRα in the regulation of these pathways suggest that increased ERRα activity may exert beneficial effects in diabetic subjects by two distinct mechanisms: enhancing mitochondrial oxidative capacity in peripheral tissues and liver, and suppressing hepatic glucose production.

Novel Mechanism for Plasma Glucose–Lowering Action of Metformin in Streptozotocin-Induced Diabetic Rats

To better understand the insulin-independent plasma glucose-lowering action of metformin, we used streptozotocin (STZ)-induced diabetic rats to investigate the possible mechanisms. Oral intake of metformin decreased the plasma glucose of STZ-induced diabetic rats with a parallel increase of plasma beta-endorphin-like immunoreactivity (BER). Mediation of opioid mu-receptors in the action of metformin was identified by the blockade of receptors with antagonist in STZ-induced diabetic rats and the failure of action in opioid mu-receptor knockout diabetic mice. Release of BER from adrenal glands by metformin was characterized, using bilateral adrenalectomy and the release of BER from isolated adrenal medulla of STZ-induced diabetic rats. Repeated treatment with metformin in STZ-induced diabetic rats increased the mRNA and protein levels of GLUT-4 in soleus muscle that was blocked by naloxonazine. Reduction of the mRNA or protein levels of hepatic PEPCK was also impeded in the same group of STZ-induced diabetic rats. In conclusion, our results provide novel mechanisms for the plasma glucose-lowering action of metformin, via an increase of beta-endorphin secretion from adrenal glands to stimulate opioid mu-receptor linkage, leading to an increase of GLUT-4 gene expression and an attenuation of hepatic PEPCK gene expression in STZ-induced diabetic rats.

Association of the promoter polymorphism −232C/G of the phosphoenolpyruvate carboxykinase gene (PCK1) with Type 2 diabetes mellitus

The phosphoenolpyruvate carboxykinase gene (PCK1) is a potential candidate gene in the pathogenesis of Type 2 diabetes mellitus. A -232C/G promoter polymorphism of PCK1 has been associated with an increased risk of Type 2 diabetes in a Canadian population. The purpose of the present study was to examine this association in a German Caucasian population. We investigated 397 subjects with Type 2 diabetes [227 men, 170 women, age 63 +/- 11 years, body mass index (BMI) 28.7 +/- 5.1 kg/m2] and 431 control subjects without diabetes (247 men, 184 women, age 64 +/- 7 years, BMI 26.5 +/- 3.7 kg/m2) matched for sex and age. In the diabetic and control groups, the CC genotype frequencies were 18.1 and 18.3%, the CG 48.6 and 48.7% and the GG 33.2 and 32.9%, respectively (P = 0.995). The allelic frequencies were 0.51 and 0.57 for the G allele and 0.49 and 0.43 for the C allele, respectively. In a logistic regression model only BMI and family history, but not the polymorphism, were predictors of Type 2 diabetes. In both the control and diabetic subjects, there were no significant differences in BMI or blood pressure between the groups with or without the polymorphism. The variant also had no significant influence on the presence of atherosclerotic disease, while the influence of other known cardiovascular risk factors was confirmed. The present data suggest that, in a German Caucasian population, the -232C/G polymorphism of the PEPCK gene is not associated with Type 2 diabetes.

Signal-dependent Control of Gluconeogenic Key Enzyme Genes through Coactivator-associated Arginine Methyltransferase 1

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.

The Lack of β-Adrenoceptors Results in Enhanced Insulin Sensitivity in Mice Exhibiting Increased Adiposity and Glucose Intolerance

We and others have previously shown that triple knockout mice lacking the beta1/beta2/beta3-adrenoceptors (beta-less mice) developed a progressive obesity at adulthood. Here, we studied the glucose homeostasis in beta-less mice before the onset of obesity. We show that beta-less mice have increased fat mass and are glucose intolerant. In addition, we observed that beta-less mice have impaired glucose-induced insulin secretion and exhibit an increase in liver PEPCK gene expression in the fed state, suggesting that they have increased gluconeogenesis. Although these characteristics are usually associated with insulin resistance, beta-less mice exhibit enhanced insulin sensitivity during insulin tolerance tests. This is keeping with the results obtained during euglycemic-hyperinsulinemic clamps showing that beta-less mice display increased insulin responsiveness with normal suppression of hepatic glucose production. Altogether, our results suggest that an intact beta-adrenergic system is required to regulate overall glucose homeostasis and, in particular, insulin-mediated glucose uptake, most likely at the level of muscles and adipose tissue.

Dual Specificity MAPK Phosphatase 3 Activates PEPCK Gene Transcription and Increases Gluconeogenesis in Rat Hepatoma Cells

Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. In insulin resistance and type II diabetes there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycemia. To search for novel genes that negatively regulate insulin signaling in controlling metabolic pathways, we screened a cDNA library derived from the white adipose tissue of ob/ob mice using a reporter system comprised of the PEPCK promoter placed upstream of the alkaline phosphatase gene. The mitogen-activated dual specificity protein kinase phosphatase 3 (MKP-3) was identified as a candidate gene that antagonized insulin suppression on PEPCK gene transcription from this screen. In this study, we showed that MKP-3 was expressed in insulin-responsive tissues and that its expression was markedly elevated in the livers of insulin-resistant obese mice. In addition, MKP-3 can activate PEPCK promoter in synergy with dexamethasone in hepatoma cells. Furthermore, ectopic expression of MKP-3 in hepatoma cells by adenoviral infection increased the expression of PEPCK and G6Pase genes and led to elevated glucose production. Taken together, our data strongly suggests that MKP-3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis. Therefore, dysregulation of MKP-3 expression and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes.

Transient impairment of the adaptive response to fasting in FXR-deficient mice

The farnesoid X receptor (FXR) has been suggested to play a role in gluconeogenesis. To determine whether FXR modulates the response to fasting in vivo, FXR-deficient (FXR −/−) and wild-type mice were submitted to fasting for 48 h. Our results demonstrate that FXR modulates the kinetics of alterations of glucose homeostasis during fasting, with FXR −/− mice displaying an early, accelerated hypoglycaemia response. Basal hepatic glucose production rate was lower in FXR −/− mice, together with a decrease in hepatic glycogen content. Moreover, hepatic PEPCK gene expression was transiently lower in FXR −/−mice after 6 h of fasting and was decreased in FXR −/−hepatocytes. FXR therefore plays an unexpected role in the control of fuel availability upon fasting.

Chronic Hyperglycemia Enhances PEPCK Gene Expression and Hepatocellular Glucose Production Via Elevated Liver Activating Protein/Liver Inhibitory Protein Ratio

Acute hyperglycemia normally suppresses hepatic glucose production (HGP) and gluconeogenic gene expression. Conversely, chronic hyperglycemia is accompanied by progressive increases in basal HGP and is a major contributor to hyperglycemia in both type 1 and type 2 diabetes by mechanisms that are poorly understood. The aim of this study was to investigate the molecular mechanisms whereby hyperglycemia contributes to excessive gluconeogenesis in Fao hepatoma cells. Increasing glucose from 5 to 20 mmol/l resulted in loss of glucose inhibition of PEPCK gene expression after 12 h. Furthermore, 24 h of incubation with 20 mmol/l glucose increased cAMP-stimulated PEPCK mRNA by approximately 40% (P < 0.05) and similarly increased glucose production. Although total CCAAT/enhancer-binding protein beta (C/EBPbeta) protein levels were suppressed, 20 mmol/l glucose increased the liver activating protein (LAP; an active isoform of C/EBPbeta)/liver inhibitory protein (LIP; an inhibitory isoform of C/EBPbeta) ratio significantly. Chromatin immunoprecipitation studies of the endogenous PEPCK gene demonstrated an increased association of LAP with the cAMP response element of the promoter. Using transient transfection to manipulate the LAP/LIP ratio, we also demonstrate a direct relationship between this ratio and PEPCK promoter activity. An increased LAP/LIP ratio not only enhanced cAMP- and dexamethasone-induced PEPCK gene expression but also impaired the repressive effect of insulin. These results demonstrate that sustained hyperglycemia diminishes the inhibitory effect of glucose and insulin on PEPCK expression and enhances hormone-stimulated PEPCK gene expression and hepatocellular glucose production. Because prolonged hyperglycemia increases the LAP/LIP ratio and can potentiate hormone induction of PEPCK transcription, our results suggest that a hyperglycemia-driven increased LAP/LIP ratio may be a critical molecular event in the pathogenesis of increased HGP in diabetes.

Elevation in Tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells.

Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.

Activating Transcription Factor-2 Mediates Transcriptional Regulation of Gluconeogenic Gene PEPCK by Retinoic Acid

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect. Furthermore, we show that RA treatment potentiates ATF-2-dependent transactivation by inducing specific phosphorylation of ATF-2 by p38β kinase. ATF-2 activation by RA blocked the inhibitory intramolecular interaction of ATF-2 amino and carboxyl terminal domains in a p38β kinase-dependent manner. Consistent with these results, RA treatment increased the DNA binding activity of ATF-2 on the PEPCK CRE-1 sequence. Taken together, the data suggest that RA activates the p38β kinase pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription and glucose production.

Activating transcription factor-2 mediates transcriptional regulation of gluconeogenic gene PEPCK by retinoic acid.

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect. Furthermore, we show that RA treatment potentiates ATF-2-dependent transactivation by inducing specific phosphorylation of ATF-2 by p38beta kinase. ATF-2 activation by RA blocked the inhibitory intramolecular interaction of ATF-2 amino and carboxyl terminal domains in a p38beta kinase-dependent manner. Consistent with these results, RA treatment increased the DNA binding activity of ATF-2 on the PEPCK CRE-1 sequence. Taken together, the data suggest that RA activates the p38beta kinase pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription and glucose production.

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.