Protein Nitration In The Tissues Of Iron Overload-type2Diabetic Rats

With the development of economy and the change of lifestyle，diabetes is taking itsplace as one of the main threats to human health in the21st century. Increasing evidencenow suggests a potential role for iron in the etiopathogenesis of type2diabetes （T2D）.However, the exact mechanism of iron-induced diabetes is uncertain. Reactive oxygenspecies （ROS） and reactive nitrogen species （RNS） induced protein post-translationalmodifications play an important role in many physiological and pathological processes.In pathological conditions, excessive production of ROS and RNS results in oxidativeand nitrative modifications of amino acid residues, altering the protein structure andfunction; thus, closely linked with the development of many diseases. Iron is a strongpro-oxidant that catalyses several cellular reactions that result in the production of ROS,with a consequent increase in the level of oxidative stress; thereby, elevating the risk ofT2D. In addition to ROS, iron can also promote the formation of RNS and catalyzereaction between the RNS and protein, causing protein tyrosine nitration. Therefore,protein tyrosine nitration may play a role in the mechanism of iron-induced diabetes. Inthis dissertation, the impacts of excessive iron on diabetic body parameters andnitrative/oxidative modifications of functional protein in diabetes were studied throughaimimal model and biochemical analysis to elucidate the relationship between iron andT2D. The main results are as follows:（1） Preliminary study on the impacts of excessive iron on diabetic body parametersMale Wistar rats were used to induce chronic iron overload and T2D byintraperitoneal injection of iron-dextran （300mg Fe/kg） and a low dose of streptozocin（STZ,35mg/kg） combined the high-fat diet （HFD）. Some basic biological indicatorswere assayed to confirm the efficacy of the induction of both iron overload and T2D.Based on this model, the growth curve, tissue/body weight ratios and liver calciumcontent were studied to preliminary discussion the influence of iron overload on T2D. The results showed that liver iron, heart iron, serum iron contents and transferinsaturation were considerably increased in the iron-supplemented rats. Additionally, bloodglucose concentrations were significantly higher in diabetic rats than that in controls, andiron supplementation did not aggravate hyperglycemia during the study. Iron supplementmarkedly increased diabetes-mediated physiological injure by decreasing body weight,increasing （liver, heart, kidney, spleen）/body weight ratio, and disturbing the calciummetabolism, causing calcium overload in the liver. The experimental results show thatiron overload can increase the adverse effects on the body of diabetic rats.（2） The role of iron induced oxidative/nitrative damage in liver dysfunction in diabeticratsExcessive tissue iron levels are associated with the increase of oxidative/nitrativestress which contributes to tissue damage that may elevate the risk of diabetes. Therefore,we investigated the effects of iron on diabetes-associated liver injury and whetheriron-related tyrosine nitration participated in this process. Iron supplement markedlyincreased diabetes-mediated hepatic dysfunction by increasing serum levels of aspartateand alanine aminotransferase, which was further confirmed by histological examination.The increased hepatic dysfunction were correlated with elevated levels of lipidperoxidation, protein carbonyls and tyrosine nitration, oxidative metabolism of nitricoxide, and reduced antioxidant capacity. Consequently, the extent of oxidized/nitratedglucokinase was markedly increased in the iron-treated diabetic rats that contribute to adecrease in its expression and activity. Further studies revealed a significant contributionof iron-induced specific glucokinase nitration sites to its inactivation. By means ofHPLC-MS/MS analysis, it was found that nitrated tyrosine residues on position Tyr-413was identified in all animal groups, and Tyr-289in HFD+STZ treated groups, whereasTyr-61was only in iron overload+diabetic group.（3） The role of iron on oxidative and nitrative modifications of sarcoplasmic reticulumCa²⁺-ATPase （SERCA2a） By means of immunoprecipitation and Western blotting analysis, the levels ofprotein carbonyls and3-nitrotyrosine in SERCA2a from different treated rat hearts weredetermine, and oxidative and nitrative modifications to protein dysfunction were alsostudied. Additionally, Effects of iron on SIN-1-induced SERCA2a dysfunction wereinvestigated in vitro. Iron supplement markedly increased diabetes-mediated cardiac totalprotein carbonylation, tyrosine nitration, and iNOS expression. And iron disturbedcalcium homeostasis, causing calcium overload in the diabetic rat hearts. Consequently,the extent of oxidized/nitrated SERCA2a was markedly increased in the iron-treateddiabetic rats that contribute to a decrease in its expression and activity. In vitroexperiments, iron markedly increased SIN-1-mediated up-regulation of SERCA2aoxidation and nitration, which were correlated with reduced SERCA2a expression andactivity. We further demonstrated that any cysteine oxidation of SERCA2a in rat heartshad minor functional effects compared with tyrosine nitration, as evidenced by the strongcorrelation between Ca²⁺-ATPase inhibition and SERCA2a tyrosine nitration in vitroconditions.