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.2023 Jan;1868(1):159247.
doi: 10.1016/j.bbalip.2022.159247. Epub 2022 Oct 20.

Lipids uniquely alter rates of insulin aggregation and lower toxicity of amyloid aggregates

Affiliations

Lipids uniquely alter rates of insulin aggregation and lower toxicity of amyloid aggregates

Mikhail Matveyenka et al. Biochim Biophys Acta Mol Cell Biol Lipids.2023 Jan.

Abstract

Amyloid formation is a hallmark of many medical diseases including diabetes type 2, Alzheimer's and Parkinson diseases. Under these pathological conditions, misfolded proteins self-assemble forming oligomers and fibrils, structurally heterogeneous aggregates that exhibit a large variety of shapes and forms. A growing body of evidence points to drastic changes in the lipid profile in organs affected by amyloidogenic diseases. In this study, we investigated the extent to which individual phospho- and sphingolipids, as well as their mixtures can impact insulin aggregation. Our results show that lipids and their mixtures uniquely alter rates of insulin aggregation simultaneously changing the secondary structure of protein aggregates that are grown in their presence. These structurally different protein-lipid aggregates impact cell viability to different extent while using distinct mechanisms of toxicity. These findings suggest that irreversible changes in lipid profiles of organs may trigger formation of toxic protein species that in turn are responsible for the onset and progression of amyloidogenic diseases.

Keywords: Atomic force microscopy; Infrared spectroscopy; Neurodegenerative disorders; Protein aggregation; Single-molecule biophysics.

Copyright © 2022 Elsevier B.V. All rights reserved.

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Conflict of interest statement

Declaration of competing interest Dmitry Kurouski reports financial support was provided by Texas A&M University. The authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.
Lipids uniquely alter the rate of insulin aggregation. Averages of triplicates of ThT aggregation kinetics of insulin in the lipid-free environment (Ins) and in the presence of CL, CER, PS, PC and SM at 1:1 molar ratio, as well as in the presence of lipid mixtures PC:CL at 1:1 and 0.8:0.2. For Ins, 400 μM of protein was dissolved in 1xPBS with 2 mM of ThT; pH adjusted to pH 3.0. For Ins:CL, Ins:CER, Ins:PS and Ins:SM, as well as for Ins:PC:CL (1:0.5:0.5) and Ins:PC:CL (1:0.8:0.2), 400 μM of insulin was mixed with an equivalent concentration of the corresponding lipid; pH was adjusted to pH 3.0. All samples were kept at 37 °C under 510 rpm for 24 h.
Fig. 2.
Fig. 2.
Lipids uniquely alter morphologies of insulin aggregates. AFM images of Ins aggregates, Ins:PS, Ins:PC, Ins:CL, Ins:PC:CL (1:0.5:0.5), Ins:PC:CL (1:0.8:0.2), Ins:SM and Ins:CER. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample aliquots were diluted with 1xPBS pH 3.0 and deposited onto pre-cleaned silicon wafer. AFM imaging was performed in tapping mode. Scale bars are 2 μm for the first and third rows and 200 nm for the second and forth rows.
Fig. 3.
Fig. 3.
Structural analysis of insulin aggregates. CD (left) and ATR-FTIR (right) spectra of insulin aggregates (Ins) grown in the lipid-free environment (red), Ins:CER (green), Ins:CL (blue), Ins:PC (gray), Ins:SM (yellow), Ins:PS (brown), Ins:PC:CL (1:0.5:0.5, navy) and Ins:PC:CL (1:0.8:0.2, purple). After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, triplicates of samples were diluted with 1xPBS pH 3.0 and placed into quartz cuvette (CD) or directly deposited onto ATR crystal (ATR-FTIR) and dried under room temperature. For each of the presented traces, three independent CD or ATR-FTIR measurements were averaged.
Fig. 4.
Fig. 4.
Nanoscale analysis of lipid content of insulin aggregates. AFM-IR spectra of insulin aggregates grown in the absence of lipids (Ins) and in the presence of PS, PC, CL, as Ins:PC:CL (1:0.5:0.5), as Ins:PC:CL (1:0.8:0.2), SM and CER. Spectra collected from individual aggregates are in gray, whereas the corresponding average spectra are in red. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample aliquots were diluted with 1xPBS pH 3.0 and deposited onto pre-cleaned silicon wafer. AFM-IR analysis was performed in contact mode. At least 30–40 individual aggregates were analyzed for each sample.
Fig. 5.
Fig. 5.
Insulin aggregates grown in the presence of lipids are less toxic than the aggregates grown in the lipid-free environment. Histograms of LDH (top), ROS (middle) and JC-1 (bottom) toxicity assays of Ins, Ins:CL, Ins:CL:PC (1:0.5:0.5), Ins:CL:PC (1:0.2:0.8), Ins:PC, Ins:PS, Ins:SM and Ins:CER, as we as CL, CL:PC (1:1), CL: PC (1:4), PC, PS, SM and CER. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample triplicates were exposed to mice midbrain N27 cells for 48 h. For each of the presented results, three independent measurements were made.
Fig. 6.
Fig. 6.
Endosomal damage induced by insulin aggregates. Representative fluorescence of images of cells exposed to insulin aggregates (top). Images are pseudo-colored green for Chmp1b and TFEB, and red for Gal3. After 24 h of incubation of insulin (400 μM) with and without lipids at 37 °C under 510 rpm, sample triplicates were exposed to HEK 293 T cells with previously transfected with Chmp1b, TFEB, and Gal3 plasmids. Scale bar is 50 μm. Histograms of fluorescent puncta per cell, as well as the sum of pixels from fluorescent puncta. For each of the presented results, at least 15 individual images were analyzed.
Scheme 1.
Scheme 1.
Mechanism of cell toxicity exerted by insulin aggregates.
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