The Mechanism Of Insulin Synthesis Dysfunction In Pancreas Induced By Oxidized Tyrosine Products

In the process of food processing and storage,there are various physical and chemical factors that can cause the oxidation of protein.In addition,the oxidized products of lipid and glycation can also cause a variety of oxidative modification of amino acid residue,lead to structural changes in protein and reduced functional properties of protein,and even cause harmful effects.In many protein oxidations,tyrosine is one of the most easily oxidized amino acids,with typical oxidative products?oxidized tyrosine products,OTPs?.The structure of OTPs is stable which is resistant to acid hydrolysis and proteases.OTPs are widely detected in meat and dairy products.The main components of OTPs include dityrosine?Dityr?,3-nitrotyrosine?3-NT?,and advanced oxidized products?AOPP?.Dityr in protein may serve as a cross-link between adjacent polypeptide chains of the same or different proteins,which is regarded as an evaluation index of protein oxidation.The former researches found the effects of dietary OTPs on the redox status.OTPs could be absorbed in the intestinal and caused a Dityr accumulation in certain tissues.However,the effects of dietary OTPs-induced oxidative stress on the energy metabolism,including the glycomatabolism,the biofunction of pancreas,and the related mechanism,is still unclear.Thus,the research on the effects of dietary OTPs and Dityr on the blood glucose and the biofunction of pancreas has theoretical and practical significance to perfect the theory of food protein,optimization of food processing technology,and improve human health.In the present study,we investigated the effects of OTPs and Dityr on the ability of insulin secretion and the related mechanisms.OTPs was produced using Cu-H₂O₂ oxidation system.Electron spin resonance?ESR?results showed that OTPs and Dityr could produce free radicals,including hydroxyl radical,superoxide radical,alkyl radical,and some unrecognized free radicals.These results demonstrated that OTPs and Dityr had a strong ability to promote oxidative stress.7-day oral toxicity test were performed in both male and female Sprague Dawley?SD?rats to asses the safety of OTPs.The results showed that the toxicology level of OTPs was non-toxic.Rats were subsequently administrated with different dose of OTPs?50,250,1250mg/kg/day?for 28 days.With the prolonged time of gavage and the increased dose of OTPs?250,1250 mg/kg/day?,the growth rate was declined significantly and the food intake increased.These findings suggest that dietary OTPs might impact the energy metabolism.In addition,the fasting blood glucose of rats treated with OTPs was markedly higher than that of control rats,and the plasma insulin level of rats treated with OTPs was significantly lower than that of control rats.OTPs administration might have the potential effects on the insulin synthesis in pancreas.The current study investigated the potential impact of dietary OTPs on the pancreatic function.SD rats were fed with OTPs at the dose of 8 g/kg diet for 6,12,24 weeks respectively.Dietary OTPs increased blood glucose and decreased plasma insulin.Furthermore,OTPs administration also caused a Dityr accumulation in pancreas of rats.In addition,OTPs induced excessive ROS production.All the changes above were time-related.A 24-week feeding experiment was performed to examine the effects of OTPs-induced oxidative damage on the biofunction of insulin synthesis in pancreas.SD rats were received standard diet together with 4 or 8g/kg diet OTPs.An antioxidant group?rats were received8g/kg diet OTPs and 2g/kg diet lipoic acid?LA??was set to strengthen our hypothesis that a diet rich in OTPs would affect insulin secretion in rats associated with impaired antioxidant capacity.After 24-week period,the body weight gains of the rats in the OTPs groups were lower than those of the rats in the control group.The daily food intake was significantly higher in the OTPs-treated rats than in the control group.LA prevented the body weight loss and appetite increase induced by OTPs.The elevated fasting blood glucose,impaired glucose tolerance,and altered mRNA level of GSIS-related genes in pancreas were detected in rats fed with OTPs.The rats fed with OTPs-containing diet also exhibited excessive ROS level in blood,accompanied by inflammation,which reflected the imbalance of redox status.In addition,the antioxidant enzymes activities of pancreas,including SOD,CAT,and GPx,were markedly lower in the OTPs-fed rats than the controls.Simultaneously,the levels of MDA,Dityr,and 3-NT significantly increased in the pancreas of OTPs-fed rats.OTPs also downregulated the mRNA level of antioxidant-related genes in the Nrf2/ARE pathway?Nrf2,GPx-1,and Ho-1?.Treatment with LA ameliorated the oxidative stress induced by OTPs.These data suggest that OTPs would induce oxidative stress and decrease antioxidant capacity of pancreas.Dityr is the main component in OTPs,an additional Dityr gavage experiment was performed to explore whether Dityr should be responsible for the injury induced by OTPs.All the changes,including increased blood glucose level,decreased insulin concentration,and impaired antioxidant capacity observed in the C57BL/6 mice after gavage were consistent with the effects induced by OTPs in the SD rats.There was no significant difference between the OTPs and Dityr treated mice.These data imply that Dityr is responsible for the pancreatic damage caused by OTPs.To clarify the mechanisms of Dityr induced oxidative stress,we performed the cell experiment using Dityr and H₂O₂.In brief,Dityr has a similar effect on oxidative stress as H₂O₂,which lead to impaired GSIS pathway.Oxidative stress could result in mitochondrial dysfunction,and the mitochondrion is crucial for ATP generation and other factors that link glucose metabolism to insulin secretion.In the current study,Dityr induced mitochondrial defects in cultured mice insulinoma MIN-6cells and mice pancreas,as evidenced by less ATP production,loss of mitochondrial membrane potential,mitochondrial DNA?mtDNA?depletion,and alteration of mRNA levels of genes involved in mitochondrial function.Moreover,the apoptosis of pancreatic islets and MIN-6 cells increased after exposure to Dityr.The findings suggested that decreased insulin secretion triggered by OTPs may be mediated by oxidative stress and mitochondrial damage in pancreatic?cells.After 10-week gavage of Dityr,mice exhibited elevated free thyroid hormones?THs?in plasma,the up-regulation of THs synthesis-specific genes expressions in thyroid glands,and the increased thyroid follicles histology shapes and areas indicated that Dityr enhanced the THs synthesis in thyroid glands.In addition,Dityr administration increased THs level in the presence of unsuppressed thyroid stimulating hormone?TSH?and m RNA level of THs synthesis related genes in hypothalamus and pituitary?Trh and Tsh?,which reflected the impaired hypothalamus-pituitary-thyroid?HPT?axis.In addition,Dityr treatment also induced energy metabolism disruption.Dityr treatment caused dysfunction of insulin synthesis in pancreas in the present of enhanced T3 level.In deed,the antagonism effect was found between Dityr and T3 in the process of glucose-stimulated insulin synthesis.The mRNA downregulation of membrane transporter of T3?MCT8?and co-activator factors?RXR?,Src-1?,together with the decreased protein level of thyroid hormone receptor?1?TR?1?in pancreas illustrated that the activation ability of T3 to downstream gene involved in insulin synthesis was suppressed by Dityr.These data suggest that Dityr suppress T3-regulated insulin synthesis stimulated by glucose via an indirect way of decreasing sensibility to T3 in pancreas.The molecular binding domain for Dityr and T3 in TR?1 is the same sites.The cell experiment using mice MIN-6 cells suggests that Dityr affect T3-stimulated protein synthesis through inhibition of the Akt phosphorylation.However,Dityr significantly suppressed the regulation effect of T3 on the expression of components related in Akt/mTOR pathway?mTOR,S6K,eIF4E,and 4E-BP1?.In this research,Dityr-induced mTOR downregulation could inhibit the phosphorylation of 4E-BP1.These observations further demonstrate that the effect of T3 on the protein synthesis is inhibited by Dityr.In conclusion,food-borne OTPs and its major constituent,Dityr,could be transported through the intestinal wall and lead to oxidative damage and inflammation,cause the dysfunction of mitochondrion in pancreatic?cells.In addition,Dityr can impact the normal activity of HPT axis,and result in the imbalance of the energy intake and expenditure.Dityr has the antagonism effect with T3 in the regulation of insulin synthesis in pancreas through suppressing the transport of T3 in pancreatic?cells,downregulation of TR?1 and its co-activator,and inhibition of Akt/mTOR pathway.The present study demonstrate that long-term dietary OTPs or Dityr is a potential risk of diabetes,and oxidative stress-induced mitochondrial dysfunction and suppressed biofunction of T3 play essential roles in OTPs/Dityr-induced functional insufficiency of pancreatic?cells.