Conclusion
Insulin ball formation and cytotoxicity may be associated with NLRP3 inflammasome activation followed by pyroptotic cell death.
Methods
Amyloid formation was evaluated by thioflavin T spectroscopic assay and scanning electron microscopy. Binding between insulin amyloid fibrils and NLRP3 was evaluated by immunoprecipitation followed by native polyacrylamide gel electrophoresis. Inflammasome activation was evaluated by immunofluorescence speck formation called "ASC speck" and Western blotting. IL-1β secretion in culture supernatants of peripheral blood mononuclear cells was evaluated by enzyme-linked immunosorbent assay. Cytotoxicity was measured by lactate dehydrogenase release assay.
Results
Insulin amyloid fibrils interact directly with NLRP3, resulting in NLRP3 inflammasome activation and pyroptotic cell death.