Hyperglycemia induces protein nonenzymeatic glycosylation in brain neurons of diabetic rats at early stage

Abstract

Background Protein nonenzymatic glycosylation is supposed to be one of mechanisms for chronic complications development in diabetes mellitus, and therefore, might play an important role in the neuronal degeneration. Objective To study the protein nonenzymatic glycosylation in brain neurons of diabetic rats, and to analyze the pathway of neuronal degeneration at the early stage of hyperglymecia. Design Randomized controlled animal experiment. Setting Department of Endocrinology, First hospital Affiliated to General Hospital of Chinese PLA and Beijing Laboratory for Brain Aging, Xuanwu Hospital Affiliated to Capital Medical University. Materials Thirty-five male Wistar rats (grade II), aged 3 months old, and 11 male purebred Kunming mice (grade III) without special pathogen, aged 3 months old, were provided by the Animal Room of Capital Medical University. Methods This experiment was carried out in the Beijing Laboratory for Brain Aging, Xuanwu Hospital Affiliated to Capital Medical University in 1998. The rats in the diabetic model group were intraperitoneally injected into 10 g/L STZ according to 60 mg/kg to establish rat models of diabetes mellitus. The blood glucose and body mass of rats in each group were determined respectively at 1, 2 and 3 months after modeling. The antibodies of advanced glycosylation end products (AGEs) of bovine serum albumin (anti-BSA) were self-prepared: ▪The antigen of AGEs-BSA was prepared. ▪Eleven male Kuming mice (grade II) of 3 months old without special pathogen were selected to inoculate AGEs-BSA. ▪ The animals were immunized. ▪Primary purification and detection of poly-antibodies of AGEs: the AGEs were performed immunohistochemical examination at 1 month after diabetic modeling by ELISA method. Main outcome measures ▪ Detection results of blood glucose and body mass of rats in two groups at different time points. ▪ Determination of polyclonal antibody titer of AGEs-BSA. ▪ Changes in immunohistochemical image of AGEs in brain tissue of rats in two groups. Results Thirteen rats in the diabetic model group and fifteen rats in the normal model group entered the stage of final analysis. ▪Changes of blood glucose and body mass: At 1, 2 and 3 months after modeling, the blood glucose of rats in the diabetic model group were respectively(28.8±2.8),(23.1±5.5),(25.4±5.1) mmol/L, which were significantly higher than those in the normal control group [(6.2±0.9),(6.1±0.8),(6.1±0.7) mmol/L, P < 0.01]; At 1, 2 and 3 months after modeling, the body mass of rats in the diabetic model group were respectively (250.1±52.2),(263.8±50.0),(261.5±42.6) g, which were significantly lower than those in the normal control group [(422.6±36.2),(462.6±39.0),(485.0±28.8) g, P < 0.01]. ▪Determination of antibody titer of immune serum: The mice were treated by AGEs-BSA of different concentrations twice. After that, the titer of AGEs -BSA was determined, and the results of which indicated that a higher absorbance existed at 1 : 1 000. ▪Determination of antigen concentration: The final titer of antibody in the abdominal dropsy was determined, and the results of which suggested that there was a much higher absorbance in the AGEs-BSA at the concentration of 5–50 mg/L. ▪Determination of antibody titer in abdominal dropsy: The antibody titer in abdominal dropsy was detected by ELISA method with antigen at 20 mg/L, which indicated that the maximum absorbance (1.265±0.039) existed at 1 : 4 000, and very larger absorbance (0.982±0.067) at 1 : 20 000. The polyclonal antibody of AGEs-BSA was successfully prepared. ?Immunohistochemical detection results: The immunohistochemical staining of AGEs showed there were positive neurons in the first month in the diabetic model group, whereas it was not significant in the normal control group. The positive substances were found mainly in the cytoplasm. Conclusion Hyperglycemia at the early stage of diabetes mellitus (1 month after modeling) can lead to protein nonenzymeatic glycosylation in brain neurons, and no obvious reactions mentioned above are found in the normal control group. It suggests that the degenerative changes of tissue structure of central nervous system are related with protein nonenzymeatic glycosylation caused by hyperglycemia.