Effects Of Alloxan And Protein Nitration On The PI(3)K Pathway In Insulin Signaling Transmission System

Because of the serious threat of diabetes mellitus to human health, some scientists pay attention to the aetiology and pathematology as well as the development of new medicine for diabetes. Animal models of Type 1 diabetes mellitus was induced by alloxan to research its pathematology and the effects of selected drugs on cutting down blood sugar. Although the investigation of the damage of alloxan to pancreaticβcells has been well reported, the effects of alloxan-induced diabetes on the mRNA expression levels of insulin signal transmission molecules in carbohydrate metabolism of rat liver and muscle have been little reported.Protein phosphorylation plays a crucial role in cellular signal transduction. In insulin signaling system, insulin transmits the signal downstream by means of autophosphorylation on tyrosine residues of insulin receptor (IR) and tyrosine phosphorylation of a family of insulin receptor substrate (IRS) proteins. Protein tyrosine nitration is an important posttranslational modification, and involves in a variety of diseases. The formation of nitrotyrosine may interfere with normal signal transduction pathways and the study of its effects on tyrosine phosphorylation is important to comprehend the function of tyrosine nitration in diabetes mellitus of insulin resistance type.In this paper, A new method with ³¹P-NMR spectroscopy has been set up to investigate in vitro phosphorylation of insulin receptor and its substrate in the PI(3)K pathway in insulin signaling transmission system.The tyrosine phosphorylation of insulin receptor and insulin receptor substrate and the effects of nitration on tyrosine phosphorylation were investigated in vitro. The effects of alloxan-induced diabetes on the mRNA expression levels of insulin signal transmission molecules in carbohydrate metabolism of rat liver and muscle were also studied. The main results obtained were as follows: (1) A new protocol with ³¹P-NMR spectroscopy has been applied to investigate in vitro autophosphorylation of insulin receptor. The results of the present study demonstrated that the time-courses or quantitative reaction of in vitro autophosphorylation of insulin receptor could be monitored by the new method. Our data illustrated that the results determined by our developed ³¹P-NMR protocol coincided with those in western-blotting analysis, which confirmed the feasibility of the established ³¹P-NMR method, and the outstanding advantage of this method is non-radioactive.(2) Three consecutive intraperitoneal injections of alloxan in Wistar rats induced diabetic model. The results of the present study demonstrated that autophosphorylation level of IR in alloxan-induced diabetic rat livers was attenuated in vitro. This alteration in the early steps of insulin signaling might be one of many insulin resistance states. Our findings confirmed that the effect of the nitration of tyrosine residue on autophosphorylation of IR was concentration-dependent of SIN-1 in vitro, and autophosphorylation level of IR was up-regulated at relatively low concentration of SIN-1 and down-regulated at relatively high concentration of SIN-1, which suggested that IR tyrosine kinase was activated at lower concentration of peroxynitrite and inactivated at higher one. Moreover, our data proved that the nitrotyrosine in the nitrated peptides mimicking the nitration of IRS1 could inhibit their insulin-stimulated tyrosine phosphorylation in vitro. Because of the electronic and steric effects of nitrotyrosine on the tyrosine residue phosphorylation, the formation of 3-nitrotyrosine might interfere with the phosphorylation reaction on phenolic hydroxyl group of tyrosine. Then, the attenuation of phosphorylation level might lead to insulin resistance in insulin signal system.(3) The thiol group amounts in liver of rats and rabbits were determined by thiol group assay. The results revealed that oxidative damage existed in liver tissue of diabetic rats and rabbits. The properties of insulin receptor in liver membrane of rats and rabbits were assayed by ¹²⁵I-insulin marking method. The optimal pH was 7.5, and it corresponded to that of blood. The experiment showed that the dissociation of IR with insulin was obviously decreased at high and low affinity sites in the liver membrane of the diabetic rat and rabbit.(4) The present study revealed that alloxan-induced diabetes had significant effects on the expression of insulin signal transmission molecules in PI(3)K pathway in rat livers by RT-PCR. Alloxan-induced diabetes attenuated the mRNA expression levels of IR, IRS1, GK and GLUT2 and the protein expression of IR in rat liver tissue, and increased the mRNA expression levels of IRS2 and PI(3)K. The hepatic glycogen content of alloxan-diabetic rats treated with insulin did not restore to the levels of normal rats under above experimental conditions. These results implied that when alloxan was used to induce diabetes rats as model animals of Type 1 diabetes mellitus, the factor of insulin resistance should be considered.(5) The effects of alloxan-induced diabetes on the mRNA expression levels of important insulin signal molecules in PI(3)K pathway in rat muscle were also studied by Real-time Quantitative Polymerase Chain Reaction. The results demonstrated that alloxan-induced diabetes attenuated the mRNA expression levels of IR, IRS1 and GLUT4 in rat muscle tissues, and increased the mRNA expression levels of PI(3)K. The decrease in mRNA expression level of GLUT4 in rat muscle tissue implied that there might be insulin resistance in insulin signal system due to the decrease in its ability of uptake glucose.