Effects Of Different Concentrations Of Insulin And Glucose On The Glycogenesis Of Retinal Müller Cells

Objective: Rabbit Müller cells are isolated and cultured in DMEM/F12 with different concentrations of glucose and insulin, just simulating hyperglycemia and hyper-insulinemia. Through periodic acid-Schiff(PAS) assay, immunofluorescence technique ,analyse the localization and semi-quantitative of glycogen, glycogen synzyme(GS) and glucose transportation protein-1(GLUT-1) in the rabbit retinal Müller cells, to observe the effect of hyperglycemia and hyper-insulinemia on the capability of transporting glucose, synthesizing and storeing glycogen. Investigate Müller cells' glycogen synthesizing, supporting nerve cells ,maintain the stable concentrations of glutamic acid of retina and so on during the hyperglycemia and hyper-insulinemia.Method: Using the explant tissue culture method, the retina obtained from adult rabbit . Cells were identified through the inverted phase contrast microscope, transmission electron microscope and immunofluorescence techniques. Through MTT assay, observe the vitality of Müller cells in the different concentrations of glucose and insulin. Using periodic acid-Schiff(PAS) assay to mark cells' glycogen, measure its average light density by the image analysis software. Using immunofluorescence techniques to mark cells' glycogen synzyme and glucose transportation protein-1 , measure its average light density by the image analysis software. Statistics analysis: datas were analysed using the Statistical Package of the SPSS11.0. Discriptive data were given as mean±SD. Continuous variable wre tested by analysis of one-way analysis of variance and during different time and concentrations varible wre tested by Two-way analysis of variance, The p-values of less than 0.05 being considered to be significant.Results: (1) The retinal tissue could be found some neurites around after 3 days,many cells could be seen around after 7 days and the entire bottle bottom was overspeads by cells after 3 weeks. Cultured cells' cytoblastema included rich 8-10nm microfilaments. More than 95% of cells were characterized by immunofluorescence staining in glia fibrillary acidic protein and cellular retinaldehyde-binding protein. (2)High glucose could stimulate Müller cells growth and GLUT-1, GS expression, strengthened Müller cells transporting glucose, synthesizing and storeing glycogen. But along with culture time extension, high glucose arrested Müller cells growth and caused Müller cells more diacaustic, some cells' neurite retracted. The OD value was less and less along with culture time extension and glucose concentration increasing. GLUT-1 expression increased but GS expression dropped after 3 days. Cells in high glucose contented more glycogen than normal group, cells in high glucose for 3days or 5days also contented more glycogen than for 1day. Part of cells in the group with 50mmol/L glucose had floated , and same of cells underneath were also fragmentized, these groups' OD value were low correspondingly, and cells contented too much glycogen , GLUT-1, GS expression decreased obviously.(3)In the 1 day group, insulin could stimulate the rabbit retinal Müller cell to up-regulate the expression of GLUT-1 and GS, and result in the significant increase intracytoplasm glycogen, especially the concentration of insulin is 4U/ml. After 3d , the insulin cuold inhibit growth of Müller cell : the increase of cell diffraction, the retraction of neurite, and many floating cells in higher concentration of insulin. The expression of GLUT-1 and GS is up-regulated in 1, 4U/ml insulin, but down-modulated in 8, 12U/ml insulin. For 5 days, the effect of insulin on retinal Müller cells is obvious : many deactivative cells in every high concentration of insulin group, however, the expression of GLUT-1 and GS still is more than normal group , but less than that of 3d. The most significant increase of intracytoplasm glycogen still are these grupes of 1, 4U/ml insulin.Conclusion: (1)Retinal Müller cells can be cultured by the explant tissue culture method, the purity of Müller cells can reach to more than 95%; (2)High glucose significantly stimulate the growth of Müller cells at early stage, upregulate the expression of GLUT-1 and GS, strengthen Müller cells transporting glucose and glycogen synthesis, to increase glycogen content of the cells; However, with the time extending , cells' growth, GLUT-1 and GS expression were inhibited, which was correlative closely with glucose concentration: the higher concentration, the stronger inhibition. Therefore, when the long-term retinal Müller cells in high glucose environment, cell growth can be affected , which can be increasing apoptosis and limit of glucose transport and glycogen synthesis; (3)Insulin also significantly stimulate the growth of Müller cells at early stage, upregulate the expression of GLUT-1 and GS, strengthen Müller cells transport glucose and glycogen synthesis, to increase glycogen content of the cells; The same to high glucose, with the time extending , cell growth, GLUT-1 and GS expression was inhibited, And there is much relative to the concentration. However, compared with the high glucose group , inhibited effect of insulin on Müller cells, growth is more obvious. After 3 days in high insulin, it can see lots of floating cells . On the one hand, Müller cells are in the decompensated state, the cells glucose intake, glycogen synthesis and glucose storage are inhibited, affecting its physiological functions which it should play. On the other hand, because insulin can inhibit the growth of Müller cells and induce cell to dye , we believe that hyperinsulinemia accelerated DR progress, is closely related to the cytotoxicity of high concentrations of insulin to retinal Müller cells.