Microwave-assisted Synthesis Of Chitooligosaccharide Guanidine And Its Effect On GLUT4 Of L6 Skeletal Muscle Cells With Insulin Resistance

Glucose transporters 4(GLUT4)are crucial to the mediation of glucose transport process.Abnormal expression of GLUTS might contribute to limitations of nutrition supplying,and thus causing diabetes.By reference to the molecular structure of metformin(an oral hypoglycemic drug),this project used chitooligosaccharide(COS)and dicyandiamide to prepare chitooligosaccharide guanidine(COSG)with different molecular weight and different degree of substitution(DS)by microwave irradiation.Comparing with metformin and insulin,the recovery effect of COS and COSG on glucose metabolism of L6 skeletal muscle cells with insulin resistance was studied.Further studies of the influence of COSG on GLUT4 and the possible signaling pathways were also explored.Firstly,COSG was prepared from COS with different molecular weight and dicyandiamide through microwave irradiation.The relationship between DS and different reaction condition such as microwave power,reaction time,pH and ratio of raw materials was discussed,respectively.GPC,IR,NMR and element analysis were employed to analyze the structure and molecular weight of the product.Results conformed that the guanidine group was introduced into chitosan main chains.The substitution degree of COSG showed a trend of decrease after increases first with the increase of microwave power.The substitution degree of COSG obtained from COS(Mr=1.5 KDa)increased and the substitution degree of COSG obtained from COS(Mw=3.0 KDa)firstly increased and then decreased with the increase of reaction time.The substitution degree of COSG decreased with the increase of pH and increased with the amount of dicyandiamide.Increases of both microwave time and pH could decline the molecular weight of COSG.Secondly,cell vitality test was conducted to examine the cell toxicity of COS and COSG of different molecular weight and different substitution degree.The insulin resistance(IR)model of L6 skeletal muscle cells was established.The influence of COS and COSG on cell vitality and glucose consumption of IR cells was observed compared with metformin and insulin.Moreover,expression and translocation of protein GLUT4 and phosphorylation level of Akt and were further investigated to explore the mechanism.Results showed that both COS and COSG were non-toxic and could improve the vitality and better promote the uptake of glucose of L6 cells compared to metformin.The effect of COSG on promoting glucose uptake was dose-dependent but had no obvious relationship with molecule weight.Metformin showed little effect on neither total expression nor membrane expression of GLUT4.COS could improve the total expression of GLUT4 protein,but no significant effect on membrane translocation of GLUT4.COSG showed little influence on total GLUT4 protein expression but significant improvement of membrane expression of GLUT4 and phosphorylation level of AKT,which was consistent with the effect of insulin.