Method for detecting whether glucose metabolism is abnormal, and prevention and treatment therefor

Abstract

A method for detecting whether glucose metabolism is abnormal comprises: detecting GPx2 gene expression, GPx2 protein expression or the activity of GPx2 protein in a test body, and making comparisons with GPx2 expression amount of a normal individual, when the GPx2 expression of the individual is significantly lower than that of the normal individual, indicating that the carbohydrate metabolism of the individual is in an abnormal state. Applications of GPx2 in the preparation of a medical composition for the treatment and prevention of type II diabetes.

Claims

1. A method for preventing or treating type II diabetes in a subject at high risk of developing type II diabetes or suffering from type II diabetes, comprising administering to the subject at high risk of developing type II diabetes or suffering from type II diabetes a composition comprising an effective amount of a plasmid comprising a nucleic acid molecule encoding glutathione peroxidase 2 (GPx2) and a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the type II diabetes is caused by hepatitis C virus infection or a high-fat diet.

3. The method of claim 1, wherein the subject is a human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows the expression level of GPx2 Log 2 protein analyzed by qPCR; 1A: the expression level of GPx2 of the abnormal glucose tolerance group; 1B: the expression level of GPx2 of the validation group.

(3) FIG. 2 shows the expression level of GPx2, PEPCK, and G6PC (G6Pase) in the livers of the mice fed with a high-fat food for 24 weeks and calculated by using the 2.sup.−Δ CT method (compared to GAPDH, presented by folds) (compared to the control group, *:p<0.05; **: p<0.01; ***: p<0.001; compared to the vehicle, ###: p<0.001).

(4) FIG. 3 shows the IPGTT results of the mice after a high-fat food; 3A: the IPGTT results of 24 weeks of high-fat food together with GPx2 over-expression plasmid; 3B: the IPGTT results, the GPx2 over-expressing plastid is administered after type II diabetes is induced by 16 weeks of high-fat food (**: p<0.01 compared to the control group; ##: p<0.01 compared to the vehicle).

(5) FIG. 4 shows the results that confirm the integrity of RNA and cDNA of the histological sections of the liver tissues from patients suffering from the hepatitis C; 4A: the RNA concentration and integrity confirmation map; 4B: the cDNA integrity confirmation map.

(6) FIG. 5 is a graph showing the results of a whole genome expression detection by a microarray chip assay.

(7) FIG. 6 shows the expression of GPx2 in HepG2, Con1, Huh7.5 cell lines.

(8) FIG. 7 shows the expression of GLUT1 and GLUT2 in the Con1 cell line administered with the GPx2 over-expressing plasmid (*: p<0.05 compared to the vehicle; **: p<0.01 compared to the vehicle; #: p<0.05 compared to the control group).

(9) FIG. 8 shows the expression of G6Pase and PEPCK in the Con1 cell administered with the GPx2 over-expressing plasmid (*: p<0.05 compared to the vehicle; **: p<0.01 compared to the vehicle; ##: p<0.01 compared to the control group; ###: p<0.001 compared to the control group).

(10) FIG. 9 shows the expression of GPx2, GLUT1 and GLUT2 in the Con1 cell lines administered with different concentrations of HCV-specific viral inhibitors (Daclatasvir) (*: p<0.05 compared to DMSO (Daclatasvir concentration 0 pM); **: p<0.01; ***: p<0.001; compared to the effect of different concentrations of Daclatasvir, #: p<0.05; ##: p<0.01).

(11) FIG. 10 shows the expression of G6PC (G6Pase) and PEPCK in the Con1 cell lines administered with different concentrations of the HCV-specific viral inhibitor (Daclatasvir) (compared to DMSO (Daclatasvir concentration is 0 pM); *: p<0.05; p<0.01; ***: p<0.001; compared to the effect of different concentrations of Daclatasvir, #: p<0.05; ##: p<0.01).

(12) FIG. 11 shows the expression of GPx2 in the liver in the mice with high-fat food for 24 weeks.

(13) FIG. 12 shows the expression of GPx2 in the liver of the mice with high-fat food for 48 weeks.

(14) FIG. 13 shows the expression of GLUT in the liver of the mice with high-fat food for 48 weeks.

(15) FIG. 14 shows the expression of the proteins related to gluconeogenesis in the liver of the mice with high-fat food for 48 weeks.

(16) FIG. 15 shows the expression of GLUT2, G6PC (G6Pase) and PEPCK after the expression of GPx2 is inhibited by GPx2 siRNA treatment.

EXAMPLES

(17) Cell Experiments

(18) Huh7.5 and Huh7.5/Con1 cell replicons (genotype 1b) were cultured in a Dulbecco's Modified Eagle's medium (DMEM) with a high concentration of glucose, incubated in a 5% carbon dioxide/37° C. incubator, and supplemented with 10% heat-inactivated fetal bovine serum (heat-inactivated FBS), 5% antibiotic-antimycotic solution, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% non-essential amino acid solution. The cell lines were first subjected to starvation treatment for 24 hours, then fresh medium containing 10% FBS and annonacin was added and in vitro experiments were performed. The Con1 cell lines were continuously cultured in a complete medium (containing 0.5 mg/mL of G418).

(19) Immunoblotting

(20) A 30 μg sample of cytolysate was placed on a 10% SDS-polyacrylamide gel, electrophoresis was performed, and transfected to a PVDF membrane. After being blocked, the membrane was separately immersed in a solution containing the target protein GPx2, GLUT1, PCK1 (PEPCK), GLUT2, and G6PC (G6Pase) antibodies (primary antibody), and incubated for two hours, and then washed with PBS containing 0.1% tween 20 for five minutes. Subsequently, the membrane was incubated in the HRP complex secondary antibody for one hour. An enhanced chemiluminescence reagent (ECL) was applied on the membrane for light irradiation to observe the protein distribution.

(21) Plasmid Construction and Transient Transfection

(22) The cells were placed in a six-well plate medium in the amount of 1.2×10.sup.5 cells per well and allowed to grow for one night. The GPx2 over-expressing plasmid and siGPx2 (GPx2 siRNA) were transfected to the cells using LipofectAMINE® at a specifically specified period of time. In addition, the vehicle was only transfected with pcDNA (a vehicle) or siNC (control siRNA) and served as the control group.

(23) Animal Experiments

(24) 24 weeks: The mice were fed with a high-fat food to induce abnormal glucose metabolism: six-week-old C57BL/6 male mice were kept in a pathogen-free environment for two weeks. All mice were then randomly divided into six groups, at least five mice in each group. Group 1 was fed with normal food; group 2 was fed with normal food, and the plasmid carrier (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected once a week via the tail vein of the mouse; group 3 was fed with normal food, and the GPx2 over-expressing plasmid (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected once a week via the tail vein of the mouse; group 4 was fed with food having 30% more fat than normal food; group 5 was with fed with a food having 30% more fat than normal food, and the plasmid carrier (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected once a week via the tail vein of the mouse; group 6 was fed with a food having 30% more fat than normal food, and the GPx2 over-expressing plasmid (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected once a week via the tail vein of the mouse. All mice were sacrificed after 24 weeks, their tissues were collected and divided into three parts: the first part was fixed with 4% formaldehyde and embedded in paraffin for a tissue section study; the second part of the tissues was stored in a tissue RNA sample preservation solution (RNAlater) and placed in an −80° C. environment for subsequent gene expression detection; the third part of the tissues was stored in liquid nitrogen.

(25) 48 weeks: Six-week-old C57BL/6 male mice were kept in a pathogen-free environment for two weeks. All mice were then randomly divided into four groups, at least five mice in each group. Group 1 was fed with normal food; group 2 was fed with a food having 30% more fat than normal food; group 3 was fed with a food having 30% more fat than normal food, and the plasmid carrier (50 μg/plasmid/mouse/week, dissolved in Turbofect) was injected once a week, 16 weeks after the mice began to be fed in the experiment, via the tail vein of each mouse; group 4 was fed with a food having 30% more fat than normal food, and the GPx2 over-expressing plasmid (50 μg/plasmid/mouse/week, dissolved inn Turbofect) was injected via the tail vein of each mouse once a week, 16 weeks after the mice began to be fed in the experiment. All mice were sacrificed after 48 weeks, their tissues were collected and divided into three parts: the first part was fixed with 4% formaldehyde and embedded in paraffin for a tissue section study; the second part of the tissues was stored in a tissue RNA sample preservation solution (RNAlater) and placed in an −80° C. environment for subsequent gene expression detection; the third part of the tissues was stored in liquid nitrogen.

(26) For those mice fed with high-fat food to induce abnormal glucose metabolism, the expression level of hepatic GPx2 was significantly decreased.

(27) For those mice feed with a high fat food and simultaneously administered with the GPx2 over-expressing plastid via the tail vein, it was found by qPCR detection that the expression of GPx2 in the liver of the mice having the GPx2 over-expressing plastid was significantly increased and the expression of the genes (PEPCK and G6PC (G6Pase)) related to gluconeogenesis was effectively decreased, as shown in FIG. 2.

(28) Glucose Tolerance Test (GTT)

(29) All mice were fasted for 12 hours and injected intraperitoneally with glucose (2 g glucose/kg body weight), followed by blood sampling from the tail at 0, 30, 60, and 120 minutes after the glucose injection. The blood glucose meter was used to detect blood glucose concentration.

(30) The results are shown in FIG. 3A, after the mice were fed with high-fat food and simultaneously administered with the GPx2 over-expressing plasmid via the tail vein for 24 weeks, glucose tolerance was tested, the mice administered with the GPx2 over-expressing plasmid had a better glucose metabolism condition. As shown in FIG. 3B, after diabetic mice, were successfully induced by a high-fat food for 16 weeks, and 32 weeks after the GPx2 over-expressing plasmids was administered via the tail vein, the results of the intraperitoneal glucose tolerance test (IPGTT) showed that GPx2 was able to improve the carbohydrate metabolism disorder effectively, and the mice administered with GPx2 over-expressing plastid showed a better carbohydrate metabolism condition, comparable to the diabetes-free mice fed with a normal food.

(31) Immunohistochemical Analysis

(32) The paraffin-embedded liver tissues were cut into 4 μm sections, microwaved at 100° C. for 30 minutes, and non-specific reactions were blocked. The sections were cultured with primary antibodies at 4° C. overnight, then rinsed twice with PBS containing 0.2% Tween 20 for 10 minutes. The sections were incubated with biotinylated secondary antibodies for one hour and then rinsed twice with PBS containing 0.2% Tween 20 for 10 minutes. Finally, the sections were stained to observe the expression of various proteins.

(33) Patient Recruitment

(34) 48 patients suffering from the hepatitis C were recruited and categorized according to each individual's oral glucose tolerance test and glycated hemoglobin (as shown in Table 1), 19 of them were patients having normal blood glucose, 11 of them were patients with abnormal glucose tolerance, and 18 of them were type II diabetes patients.

(35) TABLE-US-00001 TABLE 1 Groups of hepatitis patients Blood glucose 2 hours after oral Fasting blood administration of glycated Diagnosis glucose glucose hemoglobin Normal blood <110 mg/dL and <140 mg/dL and <5.7 glucose Abnormal glucose 110-126 mg/dL or 140-200 mg/dL or 5.7-6.5 tolerance Type II diabetes >126 mg/dL or >200 mg/dL or >6.5
Virus Typing Analysis

(36) Each patient took virus typing analysis to confirm viral genotype and virus quantitative analysis, and 5 patients with normal blood glucose, 3 patients with abnormal glucose tolerance, and 3 patients suffering from type II diabetes who had never taken insulin or oral blood glucose-lowering drugs were randomly selected as samples to perform gene chip assays to screen candidate genes, other samples were used as the validation group for verifying the candidate genes. The basic information of these two sample groups is shown in Table 2.

(37) TABLE-US-00002 TABLE 2 Normal Prediabetes Diabetes p Value Gender (Male/Female) 4/10 6/2 10/5 0.0489 Age (Standard Deviation)  57.3(13.2) 56.8(9.2) 53.9(8.2) 0.6586 BMI (Standard Deviation) 25.2(2.5) 25.3(2.8) 26.4(4.2) 0.6020 GOT (IU/L, Standard Deviation) 118.5(71.1) 103.9(32.3) 108.5(47.1) 0.8291 GPT (IU/L, Standard Deviation) 166.5(70.0) 149.4(30.3) 159.4(68.8) 0.8482 APRI (Standard Deviation)  2.0(13)  1.6(0.7)  2.1(1.5) 0.7341 0 minute-AC SUGAR (mg/dL, 84.4(8.0)  91.7(10.2) 121.8(29.9) <0.0001 Standard Deviation) 128.5(29.5) 125.3(31.5) 255.6(76.9) <0.0001 120 minutes-AC SUGAR (mg/dL, Standard Deviation) 0 minute-AC Insulin (μIU/mL,  4.9(2.9)  7.8(7.1) 18.1(5.4) 0.0300 Standard Deviation) HbA1C (%, Standard Deviation)  5.3(03)  5.8(0.1)  7.0(09) <.0001
Liver Section

(38) Liver sections of each patient were collected, RNA was extracted respectively, RNA concentrations were measured, and then individually converted to cDNA to confirm the integrity. The results are shown in FIG. 4.

(39) Whole Genome Expression Detection

(40) Whole genome expression detection was performed by using microarray chip assays to normalize all chip results, as shown in FIG. 5.

(41) Then gene expression variations were analyzed. Using a 1.5-fold variation as a threshold, genes with expression variations were selected. When patients with abnormal glucose tolerance were compared to those with normal blood glucose, the expressions of 81 genes were increased, and the expressions of 77 genes were decreased; when patients suffering from type II diabetes were compared to those with normal blood glucose, the expressions of 161 genes were increased and the expressions of 99 genes were decreased.

(42) Gene network analysis: When patients with abnormal glucose tolerance were compared to patients suffering from type II diabetes, the functions of the genes with expression variations were lipid metabolism, molecular transport, small molecule biochemistry, vitamin and mineral metabolism, etc. The results are shown in Table 3.

(43) TABLE-US-00003 TABLE 3 Description p Value Number of molecules Abnormal glucose tolerance group and normal control group Lipid metabolism 2.44E−13-6.33E−03 47 Molecular transport 2.44E−13-6.33E−03 35 Small molecule 2.44E−13-6.33E−03 52 biochemistry Vitamin and mineral 3.24E−12-5.08E−03 16 metabolism Cell growth and 2.17E−06-5.63E−03 38 proliferation Type II diabetes patients and normal control group Lipid metabolism 1.97E−23-1.00E−02 59 Small molecule 1.97E−23-1.00E−02 64 biochemistry Vitamin and mineral 1.97E−23-1.00E−02 32 metabolism Molecular transport 8.15E−09-1.00E−02 37 Nucleic acid 2.27E−08-1.00E−02 11 metabolism

(44) Significantly different candidate genes of GPx2 were obtained after the results of the patients having abnormal glucose tolerance were compared with the results of those who suffer from type II diabetes: when GPx2 was analyzed by qPCR, it was found that GPx2 varied in different degrees depending on the degree of variation in glucose tolerance, as shown in FIG. 1A. When the samples of the validation group were tested by qPCR, it showed that GPx2 still maintained significant variations, and depending on the variations in glucose tolerance, the expression level of GPx2 gene was also decreased, as shown in FIG. 1B.

(45) Gene Network Analysis

(46) Based on the predictions of the gene network analysis, GPx2 was closely related to genes related to fatty acid oxidation, glucose tolerance, glucose uptake, and gluconeogenesis.

(47) The Role of GPx2 in Carbohydrate Metabolism

(48) The role of GPx2 in carbohydrate metabolism was confirmed by cell experiments. Through inhibiting the expression of GPx2, Hepatitis C virus (HCV) allowed HCV to cause carbohydrate metabolism disorder in liver cells (reducing glucose transport and increasing gluconeogenesis), and by increasing the expression of GPx2, the abnormal glucose metabolism caused by HCV was significantly improved.

(49) Through plasmid transfection and immunoblotting experiments, it was found that the expression of GPx2 in the cells (Con1 cells) having HCV type 1b replicon-related genes was significantly decreased as compared to the cells (HepG2 and Huh7.5) without HCV-related genes, as shown in FIG. 6.

(50) Previous studies had shown that HCV was able to decrease the expression level of GLUT in the cells (Con1) having the expression of HCV related genes. Therefore, after the present invention increased the expression level of GPx2 by giving the GPx2 over-expressing plasmid, it was found that the expression of GLUT was significantly increased, indicating that GPx2 was able to improve the ability of the cells to transport glucose, as shown in FIG. 7.

(51) It had been reported that HCV was able to increase the expression level of gluconeogenesis-related genes (G6PC (G6Pase) and PEPCK) in the cells (Con1) having the expression of HCV-related genes. Therefore, after the present invention increased the expression level of GPx2 by giving GPx2 over-expressing plasmid, the expressions of G6PC (G6Pase) and PEPCK were significantly decreased to inhibit gluconeogenesis in the cells, as shown in FIG. 8.

(52) After the Con1 cells were treated with the HCV-specific viral inhibitor (Daclatasvir) and detected by Western blotting, it was found that as the concentration of the inhibitor increased, the amount of virus decreased to increase the expression of GPx2 and GLUT on concentration-dependent manner, as shown in FIG. 9. The gluconeogenesis-related proteins G6PC (G6Pase) and PEPCK were also down-regulated by Daclatasvir in a concentration-dependent manner, as shown in FIG. 10.

(53) Through the immunohistostaining results, it was found that the expression of GPx2 was decreased in high-fat food-induced diabetic mice; and after the GPx2 over-expressing plasmid was administered, the expression level of GPx2 was increased, as shown in FIG. 11. In the experiments of the 48-week high-fat food, the expression of GPx2 was also significantly increased, as shown in FIG. 12. The expression of GLUT was decreased in high-fat food-induced diabetic mice; and the expression of GLUT was increased after the GPx2 over-expressing plasmid was administered, as shown in FIG. 13. The expression level of G6PC (G6Pase) and PEPCK in high-fat food-induced diabetic mice was increased, and the expression level of G6PC (G6Pase) and PEPCK was decreased after the GPx2 over-expressing plasmid was administered, as shown in FIG. 14.