Regulation Mechanism Of Insulin Signal Transduction Pathway By Peroxynitrite

Diabetes is a common kind of metabolic diseases. Type 2 diabetes, accounting for 90% of diabetes, is characterized by peripheral insulin resistance with an insulin-secretory defect that varies in severity. Insulin resistance results from impairment of one or more steps in insulin signal transduction pathway. In the past few years, there has been accumulating evidence that RNS/ROS make a significant contribution to the progression of diabetes and its complications. Peroxynitrite (ONOO^-), derived from NO under oxidative stress, is a powerful oxidizing and nitrating species, causing DNA damage, lipid peroxidation, oxidation of protein-associated thiol groups, and nitration of protein tyrosine residues. Protein tyrosine nitration is an important posttranslational modification, and involved in a large variety of inflammatory, cardiovascular and neurodegenerative diseases. Nitration by peroxynitrite is a principal pathway, where nitrotyrosine is formed by free radical reaction. Protein tyrosine nitration can affect tyrosine phosphorylation, and thus affect signal transduction pathways involving tyrosine phosphorylation. Accumulating evidence suggests that peroxynitrite is involved in the pathogenesis of insulin resistance and diabetes, however, the regulation mechanism of insulin signal transduction pathway by peroxynitrite remains poorly understood.In this paper, the regulation mechanism of insulin signal transduction pathway by peroxynitrite was investigated in in vitro purified insulin receptor (IR), ex vivo primary skeletal muscle cells and HepG2 cells, and in vivo experimental mouse model of type 2 diabetes. The main results are as follows:(1) Effects of peroxynitrite on mRNA expression of insulin signaling molecules in primary skeletal muscle cellsTo investigate the effects of peroxynitrite treatment on gene expression profiles of insulin signaling molecules, primary skeletal muscle cells were exposed to various concentrations of peroxynitrite or 3-morpholinosydnonimine hydrochloride (SIN-1), followed by RNA isolation and real time RT-PCR analysis. The results showed that exposure of primary skeletal muscle cells to peroxynitrite (50-1000μM) led to a dose-dependent decrease in mRNA expression of IR, IRS-1, CAP, Cb1 and GLUT4. In contrast, under the same conditions, peroxynitrite had no effect on mRNA expression of PI3-K p85αand SHIP2. Moreover, exposure of primary skeletal muscle cells to SIN-1 also led to a dose-dependent decrease in mRNA expression of IR, Cbl and GLUT4. Of interest, SIN-1 treatment induced differential regulation of mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. At relatively low concentrations SIN-1 upregulated mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. However, at higher concentrations SIN-1 downregulated mRNA expression of IRS-1, CAP, PI3-K p85αand SHIP2. Taken together, these data provide evidence that high concentrations of peroxynitrite may impair insulin signal transduction in skeletal muscle cells by reducing mRNA expression of insulin signaling molecules.(2) Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cellsHepG2 cells were exposed to various concentrations of peroxynitrite, follwed by 100 nM insulin stimulation. Subcellular fractionation was performed, and thereafter protein tyrosine nitration and tyrosine phosphorylation were detected by western blotting. The results showed that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations (10-50μM) peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations (100-200μM) peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.(3) Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitriteThis report describes the effect of peroxynitrite on insulin receptor (IR) autophosphorylation and kinase activity. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, IR preparations exhibited dramatically impaired ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity with increasing peroxynitrite concentrations. Moreover, the in vivo study showed that SIN-1 administration (0.1 mg/kg and 0.25 mg/kg) decreased blood glucose levels in normal mice via upregulation of IR and IRS-1 tyrosine phosphorylation. In contrast, SIN-1 (0.25 mg/kg and 0.5 mg/kg) increased blood glucose levels in diabetic mice via downregulation of IR and IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity.(4) Peroxynitrite mediates muscle insulin resistance in mice via nitration of IRβ/IRS-1 and AktIn the current study, we investigated whether insulin resistance in vivo could be mediated by nitration of proteins involved in the early steps of the insulin signal transduction pathway. Exogenous peroxynitrite donated by 10 mg/kg SIN-1 induced in vivo nitration of the insulin receptorβsubunit (IRβ), IRS-1 and Akt, and dramatically reduced insulin signaling in skeletal muscle of mice. In high-fat diet (HFD)-fed insulin resistant mice, we observed enhanced nitration of IRβand IRS-1 and reduced insulin signaling in skeletal muscle. Reversal of nitration of these proteins by treatment with the peroxynitrite decomposition catalyst FeTPPS yielded an improvement in insulin action in skeletal muscle, in parallel with decreased nitration of IRβand IRS-1. Taken together, these findings provide new mechanistic insights for the involvement of peroxynitrite in the development of insulin resistance, and suggest that nitration of proteins involved in the early steps of insulin signal transduction is a novel molecular mechanism of HFD-induced muscle insulin resistance.(5) Preliminary study on nitroproteome in liver and skeletal muscle of diabetic miceThe nitration sites on bovine serum albumin (BSA) exposed to SIN-1 were identifiedby capillary high-performance liquid chromatography-coupled electrospray ionization tandem mass Spectrometry (LC-ESI-MS/MS). Moreover, the nitrotyrosine-containing proteins in liver and skeletal muscle from diabetic mice were investigated, and anti-nitrotyrosine immunoprecipitation products from diabetic mouse liver and skeletal muscle were analyzed by LC-ESI-MS/MS. The results showed that the nitration sites on BSA were Tyr161 and Tyr424. Moreover, protein nitration was greater in diabetic mouse liver and skeletal muscle than normal mouse liver and skeletal muscle. Sarcoplasmic reticulum Ca²⁺-ATPase was identified as one of the nitrated proteins in diabetic mouse skeletal muscle.