Xanthine oxidase modulates Glutamic Acid Decarboxylase 65 isoform (GAD65) protein expression in rat pancreatic islets

Background: It is widely presumed that aberrant expression of GAD65 can sensitize beta cells to autoimmunity and moreover, several studies indicate Xanthine Oxidase (XO) plays significant role in Type-1 diabetes (T1D) pathology. The present study aimed to investigate whether XO modulates GAD65 protein expression in rat islets/pancreas in presence or absence of STZ. Methods and results: Female Wistar rats were used for pancreatic islet isolation and T1D STZ model by injecting single dose of STZ (40 mg/kg body weight). GAD65 and XO protein expression was studied by immunoblot in islets/pancreas and STZ/T1D cytokines (such as IL-1 beta, TNF-α and IFN-γ)/XO siRNA/Allopurinol (XO inhibitor)/1400W (iNOS inhibitor) were used in treatment groups. Enzymatic assay was done to measure XO activity in islets/serum/pancreas and iNOS activity in culture supernatant. STZ led to a significant increase in GAD65 expression and XO activity in islets which was reduced in presence of Allopurinol and XO siRNA. In pancreas of T1D rat model, expression of GAD65 decreased and XO increased; XO activity decreased; whereas in sera XO activity increased. Treatment of islets with T1D cytokines revealed that cytokine cocktail declined GAD65 protein levels and XO activity and intervention with iNOS inhibitor reversed the effect. During ontogeny of pancreas GAD65 expression was more in the fetal/infantile pancreas compared to adult pancreas and Allopurinol challenged pregnant rats confirmed that GAD65 expression is dependent on XO activity. Conclusion: This study for the first time clearly demonstrates that XO is positive regulator of GAD65 expression in rat islets and STZ-T1D model.