OBJECTIVESTo explore the effects of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure.

METHODTwenty-four healthy male SD rats were randomly divided into four groups (6 rats in each group) and all of them were injected intraperitoneally with solutions: group I with normal saline, group II with 400 mg/kg of D-galactosamine (D-GaLN), group III with 400 mg/kg of D-GaLN plus 50 microg/kg lipopolysaccharide(LPS), and group IV with 400 mg/kg of D-GaLN plus 500 microg/kg LPS. At 6 hours after the administration of different solutions intraperitoneally, blood samples were collected to examine blood urea nitrogen (BUN) and serum creatinine. Realtime PCR was used to study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the livers and kidneys.

RESULTSNo endotoxemia developed in group I or group II but it was evident in group III and group IV. The level of endotoxemia in group IV was higher than in group III (8.05+/-0.43, 3.50+/-2.25, P<0.05). After 6 hours of administration of LPS in group IV, hypoglycemia appeared, and blood glucose was normal in the other three groups. BUN and serum creatinine were all normal in the four groups, except that blood urea nitrogen was elevated in group IV. The mRNA of PEPCK in livers decreased gradually in all the four groups (2.54+/-1.32 vs 1.87+/-0.15 vs 0.91+/-0.13 vs 0.44+/-0.42, P<0.05). In the kidneys there was no change in the expression of PEPCK in group I and group II (0.75+/-0.03 and 0.77+/-0.04, P>0.05), but it increased in group III (0.75+/-0.03 vs 1.63+/-0.86, P<0.05), and decreased in group IV (0.75+/-0.03 vs 0.13+/-0.07, P<0.05).

CONCLUSIONDuring acute hepatic failure severe endotoxemia would damage the function of gluconeogenesis in livers and kidneys by inhibiting transcription of PEPCK and this can induce hypoglycemia.

Animals ; Endotoxemia ; metabolism ; Gluconeogenesis ; Kidney ; metabolism ; Liver ; metabolism ; Liver Failure, Acute ; metabolism ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect and mechanism of the antihyperglycemic agent metformin on the expression of phosphoenolpyruvate carboxykinase (PEPCK) gene in hepatocytes and to determine whether the effects of metformin in hepatocytes are transmitted throughout the known insulin signaling pathways.

METHODSConfluent H4IIE rat heptoma cells were cultured for 16 h with 0.1 mmol/L metformin either in absence or presence of 0.1 nmol/L insulin, and then stimulated with various agents. The expression of PEPCK gene was examined by Northern blot analysis.

RESULTSTherapeutic concentrations of metformin significantly inhibited basal PEPCK mRNA expression and also decreased cAMP and dexamethasone induced PEPCK gene expression through interaction with insulin. In the presence of insulin signaling pathway inhibitors wortmannin and UO126, metformin reduced PEPCK mRNA levels, but wortmannin blocked inhibitory regulation of insulin on PEPCK gene expression.

CONCLUSIONMetformin inhibits PEPCK gene expression via either an insulin-independent or an interacting-with-insulin manner. The results suggest that a possible mechanism by which metformin reduces gluconeogenesis could be associated with the inhibition of PEPCK gene expression.

Androstadienes ; pharmacology ; Cells, Cultured ; Gene Expression ; drug effects ; Hepatocytes ; drug effects ; enzymology ; Humans ; Hypoglycemic Agents ; pharmacology ; Metformin ; pharmacology ; Phosphatidylinositol 3-Kinases ; physiology ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; RNA, Messenger ; analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the effects of gastric bypass surgery(GBP) on hepatic phosphoenolpyruvate carboxykinase(PEPCK) mRNA expression in type 2 diabetic Goto-Kakizaki rats.

METHODSMale GK rats were randomized into three groups: gastric bypass surgery(n=10), sham operation with diet restriction(n=10), and sham operation alone(n=10). Liver specimens of GK rats were collected during the intraoperative period for self-control study and 8 weeks after surgery. Fasting blood glucose, food intake, and body weight were recorded before surgery and 1, 2, 4, 8 weeks after surgery. The expression of PEPCK mRNA was measured by real-time PCR.

RESULTSThe fasting plasma glucose level decreased from(17.6±2.1) mmol/L before surgery to(7.5±0.9) mmol/L 8 weeks after surgery in GBP group. The level of PEPCK mRNA decreased from 1.08±0.38 before surgery to 0.41±0.10 8 weeks after surgery, significantly lower than that in sham operation alone group(1.04±0.12)(P<0.01). The level of PEPCK mRNA in diet restriction group increased from 1.15±0.16 before surgery to 2.54±0.82 8 weeks after surgery(P<0.01). The expression of PEPCK mRNA in diet restriction was significantly higher than that in CBP group(P<0.01).

CONCLUSIONSGBP can significantly improve hyperglycemia in type 2 diabetic GK rat models, which may be associated with the decrease of hepatic PEPCK mRNA level.

Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; enzymology ; surgery ; Diabetes Mellitus, Type 2 ; enzymology ; surgery ; Gastric Bypass ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Liver ; enzymology ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats

OBJECTIVETo investigate the influence and significance of gastric bypass surgery on hepatic gluconeogenesis in type 2 diabetic Goto Kakizaki(GK) rats.

METHODSForty GK rats were randomly divided into Roux-en-Y gastric bypass group(group A) and sham operation group(group B). Differences in glucose tolerance experiment(OGTT) at preoperative and postoperative 1, 2 and 4 weeks were compared and weight was recorded. Glycated hemoglobin levels were measured preoperatively and 4 weeks postoperatively. The animals were sacrificed 4 weeks after surgery and liver tissues were harvested to detect the relative expression of mRNA and protein of glucose 6 phosphatase(G-6-P) and phosphoenol pyruvate kinase(PEPCK) with RT-PCR and Western blot.

RESULTSFasting blood glucose levels were 6.5, 4.9, and 4.7 mmol/L in group A, and were 10.3, 10.4, and 12.5 mmol/L in group B, and the differences between two groups were statistically significant(P<0.05). The blood glucose level at 2 h after stomach lavage were 8.3, 6.4 and 5.5 mmol/L in group A, and were 21.4, 23.8 and 24.7 mmol/L in group B at postoperative 1, 2, 4 weeks, and the differences between two groups were statistically significant(P<0.05). The glycosylated hemoglobin at postoperative 4 weeks was(6.8±1.0)%, significantly lower than that in group B[(7.9±0.8)%, P<0.05]. Hepatic G-6-P and PEPCK mRNA relative expression at postoperative 4 weeks was reduced by 21.0% and 25.9% respectively as compared to group B, and the protein expression reduced as well. Immunohistochemistry showed that hepatic glycogen sedimentary in group A increased significantly.

CONCLUSIONThe relative mRNA and protein level of key enzymes of hepatic gluconeogenesis are significantly decreased after Roux-en-Y gastric bypass surgery and hepatic gluconeogenesis is reduced, which may be a potential mechanism of the decrease of blood glucose.

Animals ; Blood Glucose ; analysis ; Diabetes Mellitus, Experimental ; metabolism ; surgery ; Diabetes Mellitus, Type 2 ; metabolism ; surgery ; Gastric Bypass ; Gluconeogenesis ; Glucose-6-Phosphatase ; metabolism ; Glycated Hemoglobin A ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver ; enzymology ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Rats

OBJECTIVEIntrauterine growth retardation (IUGR) is associated with insulin resistance in later life but the mechanism remains unclear. To explore the molecular mechanism of insulin resistance, we determined the expression of gluconeogenic enzymes as well as the expression of transcription factor which promotes gluconeogenesis in the liver of IUGR rats.

METHODSRat model of IUGR was established by maternal proteindouble ended arrowmalnutrition. Hepatic mRNA levels of the key enzymes for gluconeogenesis, PEPCK and G6Pase, and of peroxisome proliferator-activated receptor-gammacoactivator (PGC) -1alpha were measured by RT- PCR in male IUGR pup rats at 3 and 8 weeks of their lives. Hepatic PGC-1alpha protein levels were determined by Western blot.

RESULTSThe average birth weights of the IUGR group (4.97+/-0.83 g) were significantly lower than normal controls (6.54+/-0.52 g) (P<0.01). Until to 4 weeks of age, the weights of the IUGR rats increased to the control level and were higher than normal controls at 8 weeks of age (P<0.05). There were no significant differences in blood glucose and insulin concentrations between the IUGR rats and normal controls at 3 weeks of age. By 8 weeks of age, the IUGR rats showed high insulin concentrations (P<0.01) and high insulin resistance index (P<0.05) compared with the controls. Hepatic PGC-1alpha mRNA and protein levels as well as hepatic mRNA levels of PEPCK and G6Pase in IUGR rats significantly increased at 3 and 8 weeks compared with controls.

CONCLUSIONSAn increased PGC-1alpha expression may contribute to increased mRNA levels of PEPCK and G6Pase, and thus induce the development of insulin resistance in later life in IUGR rats.

Animals ; Female ; Fetal Growth Retardation ; metabolism ; Gluconeogenesis ; Glucose-6-Phosphatase ; genetics ; Insulin Resistance ; Liver ; enzymology ; Male ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; RNA, Messenger ; analysis ; RNA-Binding Proteins ; genetics ; Rats ; Rats, Wistar ; Transcription Factors ; genetics

OBJECTIVETo study the effect of insulin, cyclic adenosine monophosphate (cAMP), and dexamethasone (DEX) on 550 bp (-600 -/+ 69) fragment of phosphoenolpyruvate carboxykinase (PEPCK) gene promoter by reporter gene.

METHODSThe recombinant pGL2-PEPCK-Luc and the control plasmid pSV-beta-Galactosidase were co-transfected to rat hepatoma cell line (CBRH7919) by lipofectin. By measuring luciferase activity, we evaluated in vitro regulation of PEPCK gene promoter on reporter gene transcription.

RESULTScAMP and DEX stimulated PEPCK promoter obviously; meanwhile, they also had accumulative effects. At different physiological concentrations, insulin had a suppressive effect on PEPCK promoter, which was dose-independent.

CONCLUSIONThere is a perfect feedback mechanism for PEPCK promoter in hepatoma cell. 550 bp (-600 -/+ 69) fragment of PEPCK may be a candidate gene in the gene therapy of diabetes.

Animals ; Cell Line, Tumor ; Cyclic AMP ; pharmacology ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Enzymologic ; drug effects ; Insulin ; administration & dosage ; pharmacology ; Liver Neoplasms, Experimental ; pathology ; Luciferases ; genetics ; metabolism ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; genetics ; Rats ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo evaluate in vitro regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene promoter on gene transcription, and construct luciferase reporter plasmid pGL2-PEPCK-Luc.

METHODSA 550 bp fragment of PEPCK promoter cut from plasmid pPEPCK-int was inserted into transitional vector PBS-SK to construct a transition plasmid PBS-PEPCK. Then the recombinant luciferase reporter plasmid pGL2-PEPCK-Luc was cloned.

RESULTSRestriction enzymes and nucleotide sequence conformed that the coupling site of recombinant plasmid was correct without base mutation and deletion, and the sequence inserted was the same as data of GeneBank. The luciferase could be expressed in hepatoma cell transfected by pGL2-PEPCK-Luc.

CONCLUSIONEstablished a new means to study transcriptional regulation of PEPCK promoter.

Animals ; Base Sequence ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Liver Neoplasms ; genetics ; Luciferases ; genetics ; Molecular Sequence Data ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; metabolism ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transcription, Genetic ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo investigate the mechanism through which Liuweidihuangwan improves hepatic insulin resistance in type 2 diabetic rats.

METHODSWith LETO rats as the normal control group, OLETF rats were treated daily with or without Liuweidihuangwan. At 8, 32, and 40 weeks of the treatment, 3 rats were randomly selected from each group for histological examination of the liver tissues and for detection of phosphoenolpyruvate carboxylase kinase (PEPCK) mRNA expression using RT-PCR and insulin receptor substrate-1 (IRS-1) and IRS-2 protein expressions using Western blotting.

RESULTSCompared with LETO rats, OLETF rats showed progressive destruction of the lobular structures and hepatic steatosis in the liver over time. OLETF rats with Liuweidihuangwan treatment had basically normal lobular structure with only mild fatty degeneration in the liver. RT-PCR detection demonstrated a significantly higher PEPCK mRNA expression in untreated OLETF rats than in LETO rats (P<0.01), but a significantly lowered PEPCK expression in OLETF rats after Liuweidihuangwan dosing (P<0.01). Western blotting showed that significantly lower p-IRS-1 and p-IRS-2 protein expressions in untreated OLETF rats than those in LETO rats and treated OLTEF rats (P<0.05).

CONCLUSIONLiuweidihuangwan improves hepatic insulin resistance in OLETF rats by inhibiting the activity of gluconeogenic key enzyme (PEPCK) in the liver and enhancing IRS-1 and IRS-2 expressions in the insulin signaling pathway.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver ; drug effects ; metabolism ; pathology ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Rats ; Rats, Inbred OLETF ; Signal Transduction

OBJECTIVETo explore the effects of arecoline on hepatic insulin resistance in type 2 diabetes rats and to elucidate its possible mechanism.

METHODSForty five Wistar rats were fed with high fructose diet for 12 weeks to induce type 2 diabetic rat model. rats were randomly divided into 5 groups (n = 8): control group, model group and model group were treated with different dose (0, 0.5, 1, 5 mg/kg) of arecoline. After 4 weeks, the fasting blood glucose, blood lipid and insulin level measured , mRNA expression of liver constitutive androstane receptor (CAR), pregnane X receptor (PXR), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR), the protein expression of p-AKT and glucose transporter4 (GLUT4) were detected by Western blot.

RESULTS1.5 mg/kg arecoline could significantly decrease the level of fasting blood glucose, blood lipid, blood insulin level and liver G6Pase, PEPCK, IL-6, TNF-alpha mRNA level in type 2 diabetes rats. 1.5 mg/kg arecoline also could significantly increase CAR, PXR mRNA level and p-AKT and GLUT4 protein expression.

CONCLUSIONArecoline improved hepatic insulin resistance in type 2 diabetes rats by increasing the mRNA levels of CAR and PXR leading to the creased glucose metabolism and inflammation related genes expression.

Animals ; Arecoline ; pharmacology ; Diabetes Mellitus, Experimental ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Wistar ; Receptors, Cytoplasmic and Nuclear ; metabolism ; Receptors, Steroid ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites (TFBSs) in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32%. Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http://bfgl.anri.barc.usda.gov/tfbsConsSites.
Algorithms ; Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; genetics ; Cell Line, Tumor ; Computational Biology ; methods ; Conserved Sequence ; Electrophoretic Mobility Shift Assay ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Luciferases ; genetics ; metabolism ; Mice ; Normal Distribution ; Oligonucleotides ; genetics ; metabolism ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; Promoter Regions, Genetic ; genetics ; Protein Binding ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Regulatory Sequences, Nucleic Acid ; genetics ; Reproducibility of Results ; Sp1 Transcription Factor ; genetics ; metabolism ; Transcription Factors ; metabolism ; Transfection