Insulin amyloid fibrils interact directly with the NLRP3, resulting in inflammasome activation and pyroptotic cell death

Abstract

Introduction Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), an intracellular pattern recognition receptor, recognizes various pathogen-associated molecular pattern and/or damage-associated molecular pattern molecules to constitute inflammasome that act as an interleukin (IL)-1β processing platform. Injected insulin is reported to induce focal amyloidosis and the formation of subcutaneous lumps called insulin balls, but the formation of subcutaneous lumps and the underlying cytotoxic mechanism has not been elucidated. 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. Conclusion Insulin ball formation and cytotoxicity may be associated with NLRP3 inflammasome activation followed by pyroptotic cell death.

Inflammasome assembly is required for intracellular formation of β2-microglobulin amyloid fibrils, leading to IL-1β secretion

“…Because we reported previously that IAPP, Aβ, and insulin amyloid fibrils interact directly with the NLRP3 inflammasome, 20 – 22 we thought that B2M might also interact with NLRP3 to support formation of amyloid fibrils. As expected, we observed a high ALPHA signal for NLRP3-Btn plus B2M amyloid fibrils ( Figure 2(b) ).…”

“…In addition, we reported that Aβ, IAPP, and insulin amyloid fibrils interact directly with NLRP3 and activate the NLRP3 inflammasome. 20 – 22 A recent report suggests that B2M acts as a driver that initiates inflammatory responses by myeloma-associated macrophages, the process of which depends on activation of the NLRP3 inflammasome after B2M accumulation in these cells. 23 …”

“…In addition, we reported that Aβ, IAPP, and insulin amyloid fibrils interact directly with NLRP3 and activate the NLRP3 inflammasome. [20][21][22] A recent report suggests that B2M acts as a driver that initiates inflammatory responses by myeloma-associated macrophages, the process of which depends on activation of the NLRP3 inflammasome after B2M accumulation in these cells. 23 These facts prompted us to hypothesize that B2M amyloid fibrils interact directly with NLRP3 and activate the inflammasome, and that low intracellular concentrations of B2M amyloid fibrils activate the NLRP3 inflammasome, thereby triggering a chain reaction of B2M amyloid fibril formation.…”

Formation of β-structures and fibrillar associates by NovoRapid insulin molecules in the presence of Saccharomyces cerevisiae yeast cell walls

“…Нарушения синтеза и биогенеза инсулина приводят к развитию диабета и необходимости для пациентов прибегать к регулярным и многочисленным инъекциям препаратов инсулина. Известно, что инсулин может агрегировать с образованием фибриллярных структур [1][2][3][4][5][6], в том числе в местах инъекций [1,7,8], образуя амилоиды и вызывая такую патологию как инъекционный инсулиновый амилоидоз, в значительной степени снижающий эффективность терапии [1]. Следует отметить, что способность фибриллизоваться in vitro описана в литературе для разных типов инсулина [1][2][3][4][5][6].…”

“…Известно, что инсулин может агрегировать с образованием фибриллярных структур [1][2][3][4][5][6], в том числе в местах инъекций [1,7,8], образуя амилоиды и вызывая такую патологию как инъекционный инсулиновый амилоидоз, в значительной степени снижающий эффективность терапии [1]. Следует отметить, что способность фибриллизоваться in vitro описана в литературе для разных типов инсулина [1][2][3][4][5][6]. Однако условия, в которых авторы этих работ проводили инкубацию, такие как рН среды, длительность и температура, не дают возможности ответить на вопрос каковы механизмы фибриллизации инсулина после инъекций in vivo.…”