Phosphoenolpyruvate Carboxykinase 1 Expression Research Articles

PCK1 Regulates Glycolysis and Tumor Progression in Clear Cell Renal Cell Carcinoma Through LDHA.

BackgroundSuppressed gluconeogenesis and increased glycolysis are common in clear cell renal cell carcinoma (ccRCC). Phosphoenolpyruvate carboxykinase 1 (PCK1) is a rate-limiting gluconeogenesis enzyme. However, the role of PCK1 in tumor metabolism and progression remains unclear.MethodsArtificial modulation of PCK1 (down- and upregulation) in two ccRCC cell lines was performed to explore the role of PCK1 in the glycolytic phenotype and in tumor growth and metastasis in vitro and in vivo. Sixty-two patients with ccRCC underwent 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography. The levels of PCK1 and lactate dehydrogenase A (LDHA) in ccRCC tissues and peritumor tissues were investigated with immunohistochemistry. The relationships between 18F-FDG accumulation and the expression of PCK1 and LDHA were analyzed. The mechanisms underlying the regulation of LDHA by PCK1 were analyzed using in vitro molecular techniques.ResultsPCK1 suppressed ccRCC cell growth and metastasis in vitro and inhibited tumorigenesis in nude mice by blocking the aerobic glycolysis pathway. Clinically, low levels of PCK1 expression were associated with poor prognosis in patients with ccRCC. The expression level of PCK1 was negatively correlated with tumor progression, the LDHA expression level and 18F-FDG accumulation in primary ccRCC tissue. We also demonstrated that PCK1 reduces the stability of LDHA through posttranslational regulation. Finally, we showed that the effects of PCK1 on glucose metabolism, cell proliferation and metastasis are mediated via the inhibition of LDHA.ConclusionOur study identified a novel molecular mechanism underlying the Warburg effect. PCK1 may serve as a candidate prognostic biomarker, and targeting the PCK1/LDHA pathway might be a new strategy to selectively inhibit tumor metabolism in human ccRCC.

Rev-erbα activation down-regulates hepatic Pck1 enzyme to lower plasma glucose in mice

REV-ERBα (NR1D1) is a nuclear heme receptor that controls many cellular processes including cell differentiation, lipid metabolism, and inflammatory responses. Although REV-ERBα has been also implicated in regulation of glucose homeostasis, the mechanism for this regulation remains unclear. Here we investigate a potential role of REV-ERBα in regulation of PCK1 (phosphoenolpyruvate carboxykinase 1), a rate-limiting enzyme in gluconeogenesis. Hepatoma cells (Hepa-1c1c7 and HepG2 cells), wild-type mice and streptozotocin-induced diabetic mice were treated with SR9009, a specific REV-ERBα agonist. The relative mRNA and protein levels of enzymes in the cells or mouse livers were determined by qPCR and Western blotting, respectively. The fasting plasma glucose test was performed to determine the effects of Rev-erbα on glucose homeostasis. Transcriptional regulation of Pck1 by Rev-erbα was investigated using a combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. SR9009 treatment significantly decreased the mRNA level of Pck1 in mouse hepatoma Hepa-1c1c7 cells, whereas other major enzymes involved in gluconeogenesis (pyruvate carboxylase, glucose-6-phosphatase, fructose bisphosphatase and Pck2) and in glycolysis (phosphofructokinase and hexokinase-1) were unaffected. Consistent with the mRNA change, the protein level of Pck1 was down-regulated. Similarly, a repressive action of REV-ERBα on PCK1 expression was observed in human HepG2 hepatoma cells. SR9009 administration to wild-type or diabetic mice significantly reduced the level of fasting plasma glucose. This coincided with decreased mRNA and protein levels of Pck1 in the liver. In addition, the diabetic mice showed an improvement in glucose tolerability after SR9009 treatment. Promoter analysis, mobility shift, and ChIP assays revealed that Rev-erbα trans-repressed Pck1 through direct binding to -325 to -320 bp region (a RevRE site) in the gene promoter. In conclusion, Rev-erbα activation down-regulates hepatic Pck1 to lower plasma glucose.

Contribution of Hepatic Retinaldehyde Dehydrogenase Induction to Impairment of Glucose Metabolism by High-Fat-Diet Feeding in C57BL/6J Mice.

Obesity and insulin resistance are associated with overexpression of retinaldehyde dehydrogenase 1 (RALDH1). We aimed to investigate the roles of hepatic RALDH1 induction in glucose metabolism impairment using mice fed with high-fat-diet (HFD). Mice were fed with HFD for 8 weeks and treated with RALDH inhibitor citral for another 4 weeks. Oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT) and insulin tolerance test were performed. Expressions of phosphoenolpyruvate carboxykinase 1 (PCK1), glucokinase (GCK) and RALDH1 were measured. Therapeutic effects of citral were also documented in diabetic rats. Effects of retinaldehyde on PCK1 and GCK expressions were examined in rat primary hepatocytes and HepG2 cells. The results showed that HFD mice were characterized by hyperlipidaemia and insulin resistance, accompanied by significantly increased RALDH1 activity and expression. Citral (10 and 50 mg/kg) ameliorated HFD-induced hyperlipidaemia and insulin resistance, as demonstrated by the improved fasting glucose, insulin levels and lipid profiles. OGTT and PTT demonstrated that citral reversed HFD-induced glucose disposal impairment and glucose production enhancement. Citral also reversed the increased PCK1 expression and decreased GCK expression by HFD. Citral therapeutic effects were reconfirmed in diabetic rats. In vitro data indicated that retinaldehyde had the strongest PCK1 induction in primary hepatocytes of diabetic rats compared with HFD rats and control rats, in line with the increased RALDH1 expression. Citral reversed the retinaldehyde-induced PCK1 expression in primary rat hepatocytes and HepG2 cells. In conclusion, RALDH1 induction impaired glucose metabolism partly via modulating PCK1 and GCK expressions. Citral improved glucose metabolism through inhibiting RALDH activity.

The role of fructose‑1,6‑bisphosphatase 1 in abnormal development of ovarian follicles caused by high testosterone concentration.

The present study aimed to identify the molecular mechanisms underlying the effects of the fructose-1,6-bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism-induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca-interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1×10−5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist-flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription-quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14-day-cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator-activated receptor γ coactivator-1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose-6-phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism-induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.

PCK1 is negatively regulated by bta-miR-26a, and a single-nucleotide polymorphism in the 3' untranslated region is involved in semen quality and longevity of Holstein bulls.

Phosphoenolpyruvate carboxykinase 1 (PCK1) is a multi-functional enzyme that plays important roles in physiological processes, including reproduction. We previously reported that the PCK1 transcript has five splice variants; PCK1-AS4, which lacks exon 5, is enriched in the testis of Holstein bulls. In the present study, we profiled select PCK1 transcript variants in the testis, epididymus, and semen of high- and low-performance bulls, and examined the possibility that microRNAs may be involved in single nucleotide polymorphism (SNP)-mediated modulation of PCK1 expression. PCK1-AS4 abundance is not significantly different between high- and low-performance bulls. Luciferase reporter assays, however, showed that bovine PCK1 expression is repressed by bta-miR-26a in HepG2 hepatocyte cells. One SNP (c. + 2183 G > T) at the miRNA-binding site of PCK1 does not influence PCK1 expression, but is associated with elevated ejaculation volume, fresh sperm motility, and genomic estimated breeding value of longevity, as well as with reduced values of composite index and calving ease. Collectively, the identified 3'-untranslated-region SNP variant highlights the importance of PCK1 in the fecundity of Holstein bulls, and implicates a role for bta-miR-26a in regulating PCK1 abundance. Further study is needed to assess the effects of other genetic variants in 5'-flanking region and exons of PCK1 on enzyme levels in the testis and sperm. Mol. Reprod. Dev. 83: 217-225, 2016. © 2016 Wiley Periodicals, Inc.

Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase‐1 expression in fetal baboon hepatocytes in vitro

AbstractBackgroundThe objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase‐1 (PEPCK‐1) is upregulated in hepatocytes isolated from fetuses of nutrient‐restricted mothers (MNR) compared with ad libitum‐fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK‐1 expression.MethodsHepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK‐1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively.ResultsFetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK‐1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK‐1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes.ConclusionsCultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.

Novel splice variants of the bovine PCK1 gene

Phosphoenolpyruvate carboxykinase 1 (PCK1), also named PEPCK-C, is a multiple-function gene that is involved in gluconeogenesis, glyceroneogenesis, reproduction, female fertility, and development of obesity and diabetes. How its many functions are regulated was largely unknown. Therefore, we investigated mRNA expression and possible splice variants of PCK1 by screening cDNA in nine tissues from Holstein bulls and cows. PCK1 mRNA was highly expressed in the liver, kidney, ovary and testis; expression levels were low in the heart, spleen, and lung tissues. Expression of this gene was not detected in skeletal muscle. This led to the discovery of five novel bovine splice variants, named PCK1-AS1-PCK1-AS5. In PCK1-AS1, 51 nucleotides in the interior of exon 2 were spliced out. In PCK1-AS2, exons 2 and 3 were altered by the alternative 3' and 5' splice sites, respectively. PCK1-AS3 was truncated from the 3' end of exon 2 to the 5' end of exon 4. In PCK1-AS4, exon 5 was completely spliced out. In PCK1-AS5, exons 5 and 6 and the 5' end of exon 7 were spliced out. These splice variants (PCK1-AS1-PCK1-AS5) potentially encoded shorter proteins (605, 546, 373, 246 and 274 amino acids, respectively), when compared to the complete protein (622 amino acids). Considering the functional domains of the PCK1 protein, it is likely that these splice variants considerably affect the function of this protein; alternative splicing could be one of the mechanisms by which the diverse functions of PCK1 are regulated.

S-Adenosylmethionine-dependent Protein Methylation Is Required for Expression of Selenoprotein P and Gluconeogenic Enzymes in HepG2 Human Hepatocytes

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.