Copper Catalyzes Tyrosine Nitration Of Insulin Receptor Kinase Domain And The Effects Of Several Flavonoids

Insulin Receptor?IR?plays an important role in the process of insulin's hypoglycemic physiological function.When insulin binds to the insulin receptor located on the cell,it causes a change in the conformation of the insulin receptor and causes insulin receptor tyrosine phosphorylation,which in turn stimulates a series of downstream signaling pathways that ultimately allow insulin to function.Phosphorylation of 1158,1162 and1163 tyrosine(Tyr^{1158/1162/1163})on the insulin receptor beta subunit determines its tyrosine kinase activity;when tyrosine phosphorylation at this site is blocked,it is extremely influences the transmission of insulin signals,causing insulin resistance.Tyrosine is also an amino acid that is prone to nitration.Copper ions can generate reactive oxygen species?ROS?and reactive nitrogen species?RNS?to induce nitration of protein tyrosine.Studies have shown that in patients with type 2 diabetes?T2D?,free copper ions are found to be significantly elevated and accompanied by insulin receptor tyrosine nitration.Protein nitration and phosphorylation are a competitive reaction.Thus,copper ions and protein tyrosine nitration may play an important role in insulin resistance in type 2 diabetes.To demonstrate this hypothesis,we selected a fragment?1126-1165?KK-1,which is closely related to insulin signaling in the insulin receptor?subunit tyrosine kinase domain,and showed that copper ions bind to KK-1.The complex,and in the presence of both hydrogen peroxide and nitrite,can mediate nitration of KK-1 tyrosine,which hinders phosphorylation of KK-1 when it is nitrated.Flavonoids can be used to treat type 2 diabetes because of their good antioxidant activity,free radical scavenging ability and ability to complex copper ions.Flavonoids can also exhibit pro-oxidative properties in the presence of transition metal ions.We selected five flavonoids:Luteolin?Lut?,Kaempferol?Kae?,Apigenin?Api?,Diosmetin?Dio?,and Genistein?Gen?to study their copper-mediated insulin resistance.The role played by the results showed that the 40?M flavonoids could completely inhibit the copper ion-induced KK-1 tyrosine nitration,but could not restore its phosphorylation modification.Although40?M flavonoids can completely inhibit the nitration of KK-1 tyrosine mediated by Cu²⁺/H₂O₂/NaNO₂ system,it cannot completely inhibit the hydroxyl radicals?·OH?induced by Cu²⁺/H₂O₂ system,hydroxyl radicals?·OH?can oxidize tyrosine to form a tyrosine free radical?·Tyr?,which in turn produces a di-tyrosine.This process inhibits the nitration of KK-1 tyrosine mediated by Cu²⁺/H₂O₂/NaNO₂ system on the one hand and phosphorylation of KK-1 on the other hand.Our study gave a relationship between copper ion,protein tyrosine nitration and insulin resistance,and revealed the role of flavonoid antioxidant and pro-oxidative properties in copper ion-mediated insulin receptor dysfunction.