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*

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*

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*

BACKGROUND/OBJECTIVES: Recently, anthocyanins have been reported to have various biological activities. Furthermore, anthocyanin-rich purple corn extract (PCE) ameliorated insulin resistance and reduced diabetes-associated mesanginal fibrosis and inflammation, suggesting that it may have benefits for the prevention of diabetes and diabetes complications. In this study, we determined the anthocyanins and non-anthocyanin component of PCE by HPLC-ESI-MS and investigated its anti-diabetic activity and mechanisms using C57BL/KsJ db/db mice. MATERIALS/METHODS: The db/db mice were divided into four groups: diabetic control group (DC), 10 or 50 mg/kg PCE (PCE 10 or PCE 50), or 10 mg/kg pinitol (pinitol 10) and treated with drugs once per day for 8 weeks. During the experiment, body weight and blood glucose levels were measured every week. At the end of treatment, we measured several diabetic parameters. RESULTS: Compared to the DC group, Fasting blood glucose levels were 68% lower in PCE 50 group and 51% lower in the pinitol 10 group. Furthermore, the PCE 50 group showed 2- fold increased C-peptide and adiponectin levels and 20% decreased HbA1c levels, than in the DC group. In pancreatic islets morphology, the PCE- or pinitol-treated mice showed significant prevention of pancreatic beta-cell damage and higher insulin content. Microarray analyses results indicating that gene and protein expressions associated with glycolysis and fatty acid metabolism in liver and fat tissues. In addition, purple corn extract increased the phosphorylation of AMP-activated protein kinase (AMPK) and decreased phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6pase) genes in liver, and also increased glucose transporter 4 (GLUT4) expressions in skeletal muscle. CONCLUSIONS: Our results suggested that PCE exerted anti-diabetic effects through protection of pancreatic beta-cells, increase of insulin secretion and AMPK activation in the liver of C57BL/KsJ db/db mice.
Adiponectin ; AMP-Activated Protein Kinases ; Animals ; Anthocyanins ; Blood Glucose ; Body Weight ; C-Peptide ; Diabetes Complications ; Fasting ; Fibrosis ; Glucose Transport Proteins, Facilitative ; Glucose-6-Phosphatase ; Glycolysis ; Inflammation ; Insulin ; Insulin Resistance ; Islets of Langerhans ; Liver ; Metabolism ; Mice* ; Muscle, Skeletal ; Phosphoenolpyruvate ; Phosphorylation ; Zea mays*

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

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

Diabetes is a global threat threatening human health in the world, with an increasing incidence rate in recent years. The disorder of glucose metabolism is one of the major factors. As relevant glucose metabolic enzymes such as alpha-glucosidase, glucose-6-phosphatase (G-6-P), glycogen phosphorylase (GP) and glycogen synthase kinase-3 (GSK-3) get involved in and control the process of glucose metabolism, the regulation of the activity of glucose metabolic enzymes is of significance to the treatment of diabetes. Traditional Chinese medicines (TCMs) have been widely researched because of their low toxicology and high efficiency, and many extracts and components from TCMs have been proven to be regulators of glucose metabolic enzymes. Compared with anti-diabetic western medicines, anti-diabetic TCMs feature safety, reliability and low price. This essay summarizes the anti-diabetic effect of TCMs on regulating glucose metabolic enzymes.
Animals ; Diabetes Mellitus ; drug therapy ; enzymology ; metabolism ; Drugs, Chinese Herbal ; analysis ; therapeutic use ; Enzyme Activators ; analysis ; therapeutic use ; Enzyme Inhibitors ; analysis ; therapeutic use ; Glucose ; metabolism ; Glucose-6-Phosphatase ; metabolism ; Glycogen Synthase Kinase 3 ; metabolism ; Humans ; Hypoglycemic Agents ; analysis ; therapeutic use ; alpha-Glucosidases ; 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 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 explore the molecular mechanism of type 2 diabetes in intrauterine growth restricted adult rats through determination of blood glucose and expression of gluconeogenic enzymes in liver.

METHODSMale intrauterine growth restriction (IUGR) offspring induced by maternal protein-malnutrition and normal controls were studied. The body weights of offspring rats were weighted from birth to 12 weeks of age. Fasting plasma glucose and insulin levels were determined by glucose oxidase method and enzyme-linked immunosorbent assay (ELISA) respectively at 1 week, 8 weeks, and 12 weeks. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) mRNA and protein levels in liver were measured by real time RT-PCR and Western blot in newborn rats (Week 1) and adult rats (Week 12).

RESULTSBirth weights of IUGR rats were significantly lower than those of controls until 4 weeks later, when IUGR rats caught up to controls. Between 8 and 12 weeks, the growth of IUGR rats surpassed that of controls. No significant differences were observed in blood glucose and insulin levels at newborn rats between the two groups. However, by the end of 8 weeks IUGR rats developed hyperinsulinemia and high insulin resistance index. At the age of 12 weeks, IUGR rats had mild fasting hyperglycemia. In addition, hepatic PGC-1 alpha mRNA and protein levels as well as hepatic mRNA levels of PEPCK and G6Pase at Week 1 and Week 12 in IUGR rats were all significantly higher than those of controls (P<0.05).

CONCLUSIONSAs a result of intrauterine malnutrition, the expression of gluconeogenic genes is exaggerated in offspring. This change stays through adulthood and may contribute to the pathogenesis of type 2 diabetes.

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