To investigate the earlier cellular alterations(Glucose-6-Pase activity and morphologic features) caused by a hepatotoxin, thioacetamide (TAA), a single dose of the agent (200mg per kg of body weight) was given intraperitoneally to mice, which were sacrificed at intervals of 4, 8 or 16 hours after corresponding treatments. For histochemical study of glucose-6-phosphatase (G6Pase) activity, unfixed frozen sections were incubation of the Wachstein and Meisel medium and stained. The smallest pieces of liver tissue were fixed in glutaraldehyde and osmic acid, and stained by the routine electron-microscopic techniques. Some pieces of liver were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. There was a rapid and progressive loss of G6Pase activity, in an orderly time sequence, in the experimental group. There were also morphologic changes: loss of cytoplasmic basophilia, cell infiltration and necrosis in the centrilobular and intermediate zones, and an increase of sER, small vesicles and ribosomes in the cytoplasm of hepatocytes, the marked changes of nuclei and nucleoli, and a slight increase of lipid droplets in the cytoplasm at 16 hours after intoxication. The correlation between these cellular alterations was discussed in view of mechanisms in the hepatotoxic action.
Acetamides/adverse effects* ; Animal ; Glucose-6-Phosphatase/metabolism* ; Liver/drug effects* ; Liver/enzymology ; Liver/ultrastructure ; Male ; Mice ; Thioacetamide/adverse effects* ; MH - ; Substances: ; Acetamides ; Thioacetamide ; Glucose-6-Phosphatase

To investigate the earlier cellular alterations(Glucose-6-Pase activity and morphologic features) caused by a hepatotoxin, thioacetamide (TAA), a single dose of the agent (200mg per kg of body weight) was given intraperitoneally to mice, which were sacrificed at intervals of 4, 8 or 16 hours after corresponding treatments. For histochemical study of glucose-6-phosphatase (G6Pase) activity, unfixed frozen sections were incubation of the Wachstein and Meisel medium and stained. The smallest pieces of liver tissue were fixed in glutaraldehyde and osmic acid, and stained by the routine electron-microscopic techniques. Some pieces of liver were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. There was a rapid and progressive loss of G6Pase activity, in an orderly time sequence, in the experimental group. There were also morphologic changes: loss of cytoplasmic basophilia, cell infiltration and necrosis in the centrilobular and intermediate zones, and an increase of sER, small vesicles and ribosomes in the cytoplasm of hepatocytes, the marked changes of nuclei and nucleoli, and a slight increase of lipid droplets in the cytoplasm at 16 hours after intoxication. The correlation between these cellular alterations was discussed in view of mechanisms in the hepatotoxic action.
Acetamides/adverse effects* ; Animal ; Glucose-6-Phosphatase/metabolism* ; Liver/drug effects* ; Liver/enzymology ; Liver/ultrastructure ; Male ; Mice ; Thioacetamide/adverse effects* ; MH - ; Substances: ; Acetamides ; Thioacetamide ; Glucose-6-Phosphatase

It is interesting and in important to study histochemical changes of glucose-6-phosphatase (G-6-Pase) activity by electron microscopy in order to promote the knowledge needed for diagnosis and prognosis in such liver diseases as von Gierke's disease, hepatoma and various other hepatocellular alterations of different origins. Since we had not accomplished the electron microscopic demonstration of G-6-Pase, although light microscopic studies on changes of the enzyme activity were done in this laboratory, this investigation was planned to obtain a satisfactory technique for ultrastructural demonstration of the enzyme activity. Unfixed frozen sections (80 micro thick) of mouse liver were washed for 2~3 minutes in a 0.4M sucrose solution (pH6.8) containing 4 mM lead nitrate and then incubated for 15~20 minutes at 32~37 degrees C in several different media to which 0.4M sucrose solution was added: A) a modification of the original Chiquoine medium, B) the first modification of the Wachstein-Meisel medium C (the second modification; the 2% lead nitrate solution was reduced in amount to 1.5 m1 instead of 3.0 ml in the medium-B). After incubation, these sections were fixed in 1% osmic acid containing sucrose, followed by embedding in Epon, ultrathin-section, mounting and staining with uranyl acetate and/or lead nitrate. By incubating the sections in the medium (B or C), satisfactory preparations were obtainable for its electron microscopic demonstration. The granular deposits of reaction products were found characteristically on the membranous component of the rough-and smooth-surfaced endoplasmic reticulum and unclear envelope. Occasional deposits were observed within cisternae or vesicles, in the nucleus, and immediate1y adjacent to the cisternal membrane and glycogen areas.
Animal ; Glucose-6-Phosphatase/metabolism* ; Liver/enzymology* ; Liver/ultrastructure ; Male ; Mice ; Microscopy, Electron

Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.
Humans ; Gene Editing ; CRISPR-Cas Systems ; Adenine ; HEK293 Cells ; Mutation ; Glucose-6-Phosphatase/metabolism*

BACKGROUND/OBJECTIVES: The goal of this study was to examine the effect of Sargassum coreanum extract (SCE) on blood glucose concentration and insulin resistance in C57BL-KsJ-db/db mice. MATERIALS/METHODS: For 6 weeks, male C57BL/KsJ-db/db mice were administrated SCE (0.5%, w/w), and rosiglitazone (0.005%, w/w). RESULTS: A supplement of the SCE for 6 weeks induced a significant reduction in blood glucose and glycosylated hemoglobin concentrations, and it improved hyperinsulinemia compared to the diabetic control db/db mice. The glucokinase activity in the hepatic glucose metabolism increased in the SCE-supplemented db/db mice, while phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities in the SCE-supplemented db/db mice were significantly lower than those in the diabetic control db/db mice. The homeostatic index of insulin resistance was lower in the SCE-supplemented db/db mice than in the diabetic control db/db mice. CONCLUSIONS: These results suggest that a supplement of the SCE lowers the blood glucose concentration by altering the hepatic glucose metabolic enzyme activities and improves insulin resistance.
Animals ; Blood Glucose ; Glucokinase ; Glucose ; Glucose-6-Phosphatase ; Hemoglobin A, Glycosylated ; Humans ; Hyperglycemia* ; Hyperinsulinism ; Insulin Resistance* ; Insulin* ; Male ; Metabolism ; Mice* ; Phosphoenolpyruvate ; Sargassum*

OBJECTIVETo investigate the expressions of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver of mice with hyperhomocysteinemia (HHcy) and explore the mechanism of gluconeogenesis induced by homocysteine.

METHODSFifty mice were randomly divided into normal control group (n=25) and HHcy group (n=25) and fed with normal food and food supplemented with 1.5% methionine, respectively. After 3 months of feeding, the fasting blood glucose and insulin levels were determined, and HOMA insulin resistance index (HOMA-IR) was calculated. The expressions of G6Pase and PEPCK in the liver of mice were detected using RT-PCR and Western blotting.

RESULTSThe fasting blood glucose and insulin levels and HOMA-IR were significantly higher in HHcy group than in the control group (P<0.05). RT-PCR and Western blotting showed that the hepatic expressions of G6Pase and PEPCK mRNA and proteins increased significantly in HHcy group compared with those in the control group (P<0.05).

CONCLUSIONHomocysteine promotes gluconeogenesis to enhance glucose output and contribute to the occurrence of insulin resistance.

Animals ; Gluconeogenesis ; Glucose-6-Phosphatase ; metabolism ; Homocysteine ; blood ; Hyperhomocysteinemia ; metabolism ; Insulin Resistance ; Liver ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Phosphoenolpyruvate Carboxykinase (ATP) ; metabolism

OBJECTIVETo explore the effect of chlorogenic acid on disordered glucose and lipid metabolism in db/db mice and its mechanism.

METHODSThirteen 5-6-week-old male db/db mice were randomly divided into db/db-CGA group (n=7) and db/db-CON group (n=6), and thirteen 5-6-week-old male db/m mice were randomly divided into db/m-CGA group (n=6) and db/m-CON group (n=7). Mice in the CGA groups were administrated with CGA 80 mg/(kg·d)by gavage, and mice in the CON groups were administrated with PBS in the same volume by gavage. Twelve weeks later, the level of biomedical parameters in plasma, liver, and skeletal muscle were determined, the concentrations of adiponectin and visfatin in visceral adipose, and the mRNA expression of glucose-6-phosphatase (G-6-Pase) and peroxisome proliferators-activated receptor-α (PPAR-α) as well as the protein level of PPAR-α in liver were detected.

RESULTSTwelve weeks after CGA administration, the levels of triglycerides in plasma, liver, and skeletal muscle and the fasting plasma glucose in db/db-CGA group were significantly lower than those in db/db-CON group(P<0.05). The muscle glycogen level was significantly higher than that in db/db-CON group (P<0.05), and the adiponectin concentration was significantly higher than that in db/db-CON group ( P<0.01) and lower than that in db/m-CGA group(P<0.05). The visfatin concentration in db/db-CGA group was significantly lower than that in db/db-CON group (P<0.01) and significantly higher than that in db/m-CGA group(P<0.05). The mRNA expression level of G-6-Pase was significantly down-regulated in db/db-CGA group when compared with db/db-CON group (P<0.05). Both the mRNA and the protein expression levels of PPAR-α were significantly up-regulated in db/db-CGA group(P<0.05) compared with in db/db-CON group.

CONCLUSIONCGA improves the disordered glucose/lipid metabolism in db/db mice, which is speculated to be related with its role in modulating the adipokines secretion, up-regulating hepatic PPAR-α, and inhibiting G-6-Pase expression.

Adiponectin ; metabolism ; Animals ; Chlorogenic Acid ; pharmacology ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Glucose ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Lipid Metabolism ; drug effects ; Liver ; metabolism ; Male ; Mice ; PPAR alpha ; metabolism

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

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

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.
Catalytic Domain ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glucose-6-Phosphatase/metabolism* ; Humans ; Liver Neoplasms/genetics*