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.

Keywords: NLRP3 inflammasome; amyloid; insulin ball; pyroptotic cell death.

Figure 1.

Insulin oligomerization and morphological changes. (a) ThT assay results showing the formation of insulin amyloid under oligomerization conditions at 65°C for 0 h (monomers), 2.5 (intermediates), or 5 h (fibrils). (b) Insulin monomers (2 mg/mL) were incubated under oligomerization conditions at 65°C for 0 h (monomers), 2.5 h (intermediates), or 5 h (fibrils) and subjected to 10% native polyacrylamide gel electrophoresis followed by Western blotting (WB) and Coomassie Brilliant Blue (CBB) staining. (c) Scanning electron microscopy (SEM) images of insulin samples under oligomerization conditions at 65°C for 0 h (monomers), 2.5 h (intermediates), or 5 h (fibrils). Scale bars = 1 μm.

Figure 2.

Insulin fibrils interact with NLRP3-LRR. (a) Schematic representation of the full-length NLRP3 structure depicting exon components and truncated forms. The pyrin domain (PYD) is indicated by black boxes, the nucleotide-binding oligomerization domain (NOD) is indicated by a light gray box, and leucine-rich repeats (LRR) are indicated by striped boxes. Amino acid sequence numbers are also indicated. (b) FLAG-tagged truncated forms of NLRP3 proteins and Aβ fibrils were precipitated by anti-Aβ mouse mAb conjugated with protein A and detected by WB with anti-FLAG mouse mAb or anti-Aβ mouse mAb. (c) Biotinylated truncated forms of NLRP3 proteins and insulin fibrils were precipitated with anti-insulin rabbit mAb conjugated with protein A followed by WB with streptavidin or anti-insulin guinea pig pAb.

Figure 3.

Insulin fibrils activate inflammasome. (a) Western blotting analysis of pro-IL-1β production (left panel) and IL-1β cleavage (center panel; the same blotting membrane shown in the upper panel was re-hybridized with a mAb specific for cleaved-IL-1β) using PBMCs incubated with 10 μM insulin amyloid fibrils together with indicated amounts of LPS. The same gel was stained with Coomassie Brilliant Blue (CBB, right panel). (b) IL-1β concentrations in the PBMCs culture supernatant of each well measured by ELISA. *p-value < 0.05 (Mann–Whitney U-test). (c) Lactate dehydrogenase (LDH) activity in the PBMCs culture supernatant of each well (A₄₉₀). *p-value < 0.05 (Mann–Whitney U-test). (d) Immunofluorescence microscopy evaluation of ASC-speck formation. Scale bars = 10 μm. (e) Percentage of ASC specks (%) in PBMCs without (upper panel) or with (lower panel) 0.1 ng/mL LPS. *p-value < 0.05 (Mann–Whitney U-test).