The Mechanism Of The Protective Effect Of Insulin Against 1-Methyl-4-phenylpyridine-Induced Apoptosis In PC12 Cells

Parkinson's disease(PD) is one of the most common neurodegenerative disorders. It is caused by the degeneration of nigro striatum dopaminergic neuron resulting in the reduction of dopamine and the hyperfunction of acetylcholine. The cause of the more prevalent idiopathic PD are still uncertain. Acquired disturbances of cell metabolism in PD are supposed to be a general cause which indicate insulin and its signal transduction may play crucial roles. In this experiment, it is l-methyl-4-phenylpyridine(MPP+) that induces apoptosis in the rat pheochromocytoma lines (PC 12 cells). PC 12 cells have been widely used for the model of PD. The effect of insulin on MPP+-induced in PC 12 cells was investigated.Part I Insulin Can Protect PC 12 cells from MPP+-Induced Apoptosis3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to observe the change of PC 12 cells viability when cells were exposed to the different concentrations and time courses of MPP+ and the insulin. The results suggested that with the increase of the concentration and time course of MPP+, the cells viability decreased; insulin could protect PC 12 cells from the cytotoxity of MPP+. The expression of the mRNA of tyrosine hydroxylase (TH mRNA ) was measured by using RT-PCR, the similar results were obtained. HOECHST 33258 staining method and flow cytometric analysis confirmed apoptosis by MPP+ and anti-apoptosis by insulin. All above demonstrated insulin can protect PC 12 cells from MPP+-induced apoptosis.Part II Increase of phosphorylation of insulin receptor may benefit to insulin against MPP induced apoptosis in PC 12 cellsOn the basis of Part II, The expression of the mRNA of insulin receptor(IR mRNA) was measured by using RT-PCR, the expression of IR protein was measured by using immunoprecipitation and Western immunoblotting method. Both did not alter. HNMPA-AM3 -the insulin receptor tyrosine kinase inhibitor was used to determine the cells viability. It suggested that the phosphorylation of IR maybe a crucial mechanism. The wild-type human IR was transiently transfected to PC 12 cells(PC121R cells). Comparing the PC12IR cells viability with PC 12 cells transfected with pcDNA3, PC12IR cells viability was enhanced again. In the different experiment conditions, the expression of the IRmRNA and the expression of IR protein did not change except for transfected PC 12 cells; in contrast, the phosphorylation of IR was modified with the alteration of the cells viability. Increase of the phosphorylation of insulin receptor may benefit to insulin against MPP+-induced apoptosis in PC 12 cells.Part III PI-3-K/PKB is an effective pathway in insulin -insulin receptor signal transduction against apoptosis of PC 12 cellsTo elucidate PI-3-K/PKB contributed to the protection of insulin against MPP+ -induced apoptosis in PC 12 cells, Wortamannin~the specific inhibitor of PI-3-K was used. It seems that once the activation of PI-3-k was blocked, the protection of insulin was almost discharged. The expression of PKB protein did not change, but the phosphorylation of PKB at Serine(Ser)473 residues altered. The phosphorylation of PKB was an important molecule against MPP+ -induced apoptosis in PC 12 cells. Insulin-insulin receptor mainly via the activation of PI-3-K/PKB protected PC 12 cells from MPP+-induced apoptosis.Conclusion: 1. MPP+exerted cytotoxity in a dose-dependent and time-course dependent manners and that administration of insulin had aprotective effect against MPP+ -induced aipoptosis. 2. In different experiment concidions, both the expression of the IRmRNA and the expression of IR protein did not alter. HNMPA-AM3 -the insulin receptor tyrosine kinase inhibitor could block the anti-apoptosis effect of insulin. Insulin could increase the viability of PC 12 cells which over -expressed insulin receptor. These result suggested that the phosphorylation of IR maybe a crucial mechanism in this phenomenon. 3. Wortamannin~the specific inhibitor of PI-3-K inhibitor, could partially block the protection of insulin. The pho...