OBJECTIVETo investigate the effects of hypoxia on the glycolysis in cultured rat hepatocytes.

METHODSMixed gas with different concentrations of O(2), CO(2) and N(2) was prepared for the in vitro culture of normal rat hepatocytes. The cell strains were set to be A, B, C groups, which were observed at 1, 2, 4, 8 and 16 hours after hypoxia with normal hepatocytes as the control. Biochemical methods were employed to determine the activities of the key enzymes during hepatocytic glycolysis such as hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and the change of the content of lactic acid (LA) in the culture fluid.

RESULTS(1) The LDH activity of the rat hepatocytes increased significantly at all the time points of hypoxia in A and B groups when compared with that in control group (P < 0.05), while the activity increased obviously in C group since 2 hours after hypoxia (P < 0.05). (2) The HK activity of the cells in A group increased significantly at 1, 2, 4 and 16 hours after hypoxia and that in B and C groups increased obviously at 1 hour when compared with control group (P < 0.05). While the cellular PFK activity in A group increased markedly at 1 and 4 hours after hypoxia and that in B and C groups increased evidently at 4 hours after hypoxia (P < 0.05). The cellular PK activity in all the three groups increased at all the hypoxic time points (P < 0.05). (3) The cellular LA content in A and B groups began to increase since 2 hours and that in C group did so since 4 hours after hypoxia and increased along with the time lapse (P < 0.05).

CONCLUSIONhypoxia might initiate glycolysis.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; Hepatocytes ; enzymology ; metabolism ; Hexokinase ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; metabolism ; Oxygen ; metabolism ; Phosphofructokinases ; metabolism ; Pyruvate Kinase ; metabolism ; Rats

OBJECTIVETo evaluate the use of homologous gene quantitative PCR (HGQ-PCR) as a method for non-invasive diagnosis of Down syndrome and for prevention of the birth of Down syndrome children.

METHODSHGQ-PCR, which can directly detect the additional copy of chromosome 21 by comparing simultaneously amplified two highly homologous genes, i.e. the human liver-type phosphofructokinase located on chromosome 21 critical region of Down syndrome (PFKL-CH21) and the human muscle-type phosphofructokinase located on chromosome 1 (PFKM-CH1), was performed in 38 clinically diagnosed Down syndrome patients and 178 normal controls.

RESULTSThe ratios of PFKM-CH1/PFKL-CH21 products were 1.40 +/- 0.367 (mean +/- SD) and 0.46 +/- 0.21 (mean +/- SD) for disomy 21 and trisomy 21, respectively. The difference between these two groups was statistically significant (P<0.001).

CONCLUSIONThis approach has proven to be a practical and direct method for the detection of trisomy 21 and may also be applied to the detection of the extra piece of 21q involved in translocation-type of Down syndrome.

Chromosomes, Human, Pair 1 ; genetics ; Chromosomes, Human, Pair 21 ; genetics ; Down Syndrome ; diagnosis ; genetics ; Female ; Humans ; Phosphofructokinases ; genetics ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Reproducibility of Results ; Sensitivity and Specificity

This study is to investigate the inhibitory effect and mechanism of prosapogenin A (PSA) on MCF7. MTT assay was performed to determine the inhibitory effect of PSA on MCF7 cells. PI/Hoechst 33342 double staining was used to detect cell apoptosis. RT-PCR was used to test the mRNA levels of STAT3, GLUT1, HK and PFKL. Western blotting was performed to determine the expression of STAT3 and pSTAT3 protein in MCF7 cells. The results showed that PSA could dose-dependently inhibit cell growth of MCF7 followed by IC50 of 9.65 micrmol x L(-1) and promote cell apoptosis of MCF7. Reduced mRNA levels of STAT3, HK and PFKL were observed in MCF7 cells treated with 5 micromol x L(-1) of PSA. PSA also decreased the level of pSTAT3 protein. STAT3 siRNA caused decrease of mRNA of GLUT1, HK and PFKL which indicated STAT3 could regulate the expressions of GLUT1, HK and PFKL. The results suggested that PSA could inhibit cell growth and promote cell apoptosis of MCF7 via inhibition of STAT3 and glycometabolism-related gene.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Glucose Transporter Type 1 ; genetics ; metabolism ; Hexokinase ; genetics ; metabolism ; Humans ; MCF-7 Cells ; Phosphofructokinases ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; RNA, Messenger ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Saponins ; isolation & purification ; pharmacology ; Veratrum ; chemistry

OBJECTIVE: To study the changes of liver phosphofructokinase-2 (PFK-2) level at different postmortem intervals as well as due to different causes of death. METHODS: One hundred and five rats were randomly divided into 3 groups and the rats were sacrificed by either bleeding, suffocating, and neck breaking, respectively. The content of liver PFK-2 at 0, 1, 2, 4, 8, 12 and 24 hours following death were studied using immunohistochemishtry and image analysis. RESULTS: PFK-2 content of the rat's liver in all 3 groups showed a linear decrease at different postmortem intervals with no significant statistical differences found between the different groups. CONCLUSION PFK-2 level in rat liver appears to decrease linearly in correlation with prolonged PMI.
Animals ; Asphyxia/metabolism* ; Cause of Death ; Cervical Vertebrae/injuries* ; Female ; Immunohistochemistry ; Liver/enzymology* ; Male ; Phosphofructokinase-2/metabolism* ; Postmortem Changes ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic/metabolism* ; Staining and Labeling ; Time Factors

As an important component of the tumor microenvironment, cancer-associated fibroblasts (CAFs) secrete energy metabolites to supply energy for tumor progression. Abnormal regulation of long noncoding RNAs (lncRNAs) is thought to contribute to glucose metabolism, but the role of lncRNAs in glycolysis in oral CAFs has not been systematically examined. In the present study, by using RNA sequencing and bioinformatics analysis, we analyzed the lncRNA/mRNA profiles of normal fibroblasts (NFs) derived from normal tissues and CAFs derived from patients with oral squamous cell carcinoma (OSCC). LncRNA H19 was identified as a key lncRNA in oral CAFs and was synchronously upregulated in both oral cancer cell lines and CAFs. Using small interfering RNA (siRNA) strategies, we determined that lncRNA H19 knockdown affected proliferation, migration, and glycolysis in oral CAFs. We found that knockdown of lncRNA H19 by siRNA suppressed the MAPK signaling pathway, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and miR-675-5p. Furthermore, the lncRNA H19/miR-675-5p/PFKFB3 axis was involved in promoting the glycolysis pathway in oral CAFs, as demonstrated by a luciferase reporter system assay and treatment with a miRNA-specific inhibitor. Our study presents a new way to understand glucose metabolism in oral CAFs, theoretically providing a novel biomarker for OSCC molecular diagnosis and a new target for antitumor therapy.
Cancer-Associated Fibroblasts/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glycolysis ; Head and Neck Neoplasms ; Humans ; MicroRNAs/metabolism* ; Mouth Neoplasms/genetics* ; Phosphofructokinase-2/genetics* ; RNA, Long Noncoding/genetics* ; Signal Transduction ; Tumor Microenvironment

OBJECTIVE: The aim of this study is to investigate the expression of phosphofructokinase 1 and it's enzyme activity in nasopharyngeal carcinoma biopsy samples. METHOD: Sixty-one biopsy samples were detected, including 41 tissues from patients with nasopharyngeal carcinoma as experimental group and 20 tissues from patients with chronic nasopharyngitis as control group. Phosphofructokinase 1 protein was detected by Western blot and it's enzyme activity was detected. RESULT: It was observed that the expression levels of phosphofructokinase 1 protein and it's enzyme activities in the experimental group were higher than that in the control group (P < 0.01). In the experimental group, the expression levels of phosphofructokinase 1 protein and it's enzyme activities in patients with lymph node metastasis were higher than that in patients without lymph node metastasis (P < 0.01). CONCLUSION Phosphofructokinase 1 may be a marker in occurrence and metastasis of nasopharyngeal carcinoma.
Biomarkers, Tumor ; metabolism ; Biopsy ; Carcinoma ; Case-Control Studies ; Humans ; Lymphatic Metastasis ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; enzymology ; pathology ; Nasopharyngitis ; Phosphofructokinase-1 ; metabolism

OBJECTIVE: The aim of this study is to investigate the expression and clinical significance of 6-phosphofructo-1-kinase in nasopharyngeal carcinoma biopsy samples. METHOD: Sixty-one biopsy samples were detected, including 41 tissues samples from patients with nasopharyngeal carcinoma and 20 tissues samples from patients with chronic nasopharyngitis as control group. 6-phosphofructo 1 kinase mRNA expression was detected by RTPCR. RESULT: It was observed that the expression levels of 6 phosphofructo-1-kinase mRNA in nasopharyngeal carcinoma tissues were higher than in the chronic inflammatory tissues. And the expression levels in patients with lymph node metastasis were higher than without lymph nod metastasis. CONCLUSION 6-phosphofructo-1-kinase may be a marker in occurrence and metastasis of nasopharyngeal carcinoma.
Biomarkers, Tumor ; metabolism ; Carcinoma ; Female ; Humans ; Lymphatic Metastasis ; Male ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Phosphofructokinase-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism

OBJECTIVETo investigate the influence of hypoxia induction factor-1alpha (HIF-1alpha) on glycosis of rat myocardial cell under hypoxic condition.

METHODSThe myocardial cells of the rats were routinely isolated and cultured. The cells were divided into single hypoxia (H) and HIF-1alpha inhibiting (I) groups. The cells in H group were cultured in glucose-free medium with mixed low-oxygen gas [1% O2, 94% N2 and 5% CO2 (v/v)]. While the cells in I group were cultured with low-oxygen gas after the cell model of low expression of HIF-1alpha protein constructed by RNAi technique. The cells in both groups were all observed before hypoxia (routine culture) and at the time points of 1, 3, 6, 12 and 24 hours of hypoxia. The LA (lactate acid ) content in the supernatant of the culture and the activity of the key enzymes in glycolysis such as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) of both groups of cells were determined at all the time points.

RESULTS(1) After hypoxia, the HK and PFK activities of the rat myocardial cells in H and I groups were obviously increased at the beginning and decreased thereafter when compared with that before hypoxia. While the activities of HK and PFK in H group at 1, 3 and 6 hours after hypoxia were evidently higher than those in I group (P <0.05 or 0.01), and the peak activity of them in H and I groups was 159 +/- 13 U/g vs 133 +/- 55 U/g, and 298 +/- 44 U/g vs 188 +/- 55 U/g, respectively. (2) Compared with normal control (92 +/- 12 U/g), the LDH activity of the cells in H group after hypoxia increased significantly, reaching the peak at 6 hours after hypoxia (2 568 +/- 125 U/g, P < 0. 01), and it decreased thereafter, while that in I group peaked at 3 hours after hypoxia (2125 +/- 126 U/g, P <0.01). The LA content in the culture supernatant in H group increased significantly after hypoxia with the passage of time, while that in I group increased in smaller magnitude (P <0.01).

CONCLUSIONHigh expression of HIF-1alpha in the rat myocardial cells after hypoxia could directly cause continuous enhancement of cell glycolysis, which was beneficial to the protection of myocardial cells under hypoxic condition.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; Hexokinase ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; metabolism ; Phosphofructokinase-1 ; metabolism ; RNA Interference ; Rats ; Rats, Sprague-Dawley

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB-treated group and 36.7% decrease in 1 mM 3-AB-treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.
Animals ; Cell Death ; Epithelial Cells* ; Glucose ; Glucose-6-Phosphate Isomerase ; Glycolysis ; Hexokinase ; Kidney* ; LLC-PK1 Cells ; Oxidoreductases ; Phosphofructokinase-1 ; Phosphopyruvate Hydratase ; Poly Adenosine Diphosphate Ribose* ; Poly(ADP-ribose) Polymerases* ; Pyruvate Kinase ; Swine

This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.
Bioreactors ; microbiology ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Glycerol ; metabolism ; Glycolysis ; Hexokinase ; metabolism ; Industrial Microbiology ; methods ; Phosphofructokinase-1 ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; metabolism ; Schizosaccharomyces ; enzymology ; genetics ; metabolism ; Trehalose ; metabolism ; Triticum ; metabolism ; Zea mays ; metabolism