Long-Chain Polyunsaturated Fatty Acids Accelerate the Rate of Insulin Aggregation and Enhance Toxicity of Insulin Aggregates

Abstract

Long-chain polyunsaturated fatty acids (LCPUFAs) are essential components of a human diet. These molecules are critically important for cognitive attention and memory, mood states, coronary circulation, and cirrhosis. However, recently reported findings demonstrated that docosahexaenoic (DHA) and arachidonic acids (ARA), ω-3 and ω-6 LCPUFAs, accelerated the aggregation rates of insulin and α-synuclein, proteins that are directly linked to diabetes type 2 and Parkinson's disease, respectively. Furthermore, both DHA and ARA uniquely altered the structure and toxicity of the corresponding protein aggregates. Our objective is to ascertain whether other LCPUFAs, alongside long-chain unsaturated fatty acid (LCUFA) proteins, exhibit similar effects on amyloidogenic proteins. To explore this matter, we investigated the effect of 10 different LCPUFAs and LCUFAs on the rate of insulin aggregation. We found that all of the analyzed fatty acids strongly accelerated insulin aggregation. Moreover, we found that protein aggregates that were formed in the presence of these fatty acids exerted significantly higher cell toxicity compared with insulin fibrils grown in the lipid-free environment. These findings show that interactions between amyloid-associated proteins and LCPUFAs can be the underlying molecular cause of neurodegenerative diseases.

Keywords: LCPUFAs; LCUFAs; fibrils; insulin; oligomers; toxicity.

Figure 1

LCUFAs uniquely alter the aggregation rate of insulin. ThT aggregation kinetics (a) with a histogram (b) that summarizes the mean values (±SEM) of t_lag, t_1/2, and t_grow of insulin in the lipid-free environment (gray), as well as in the presence of C16:1 (yellow), C18:0 (green), trans C18:1 (orange), and cis C18:1 (red). P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001. “NS” nonsignificant difference. Each kinetic curve is the average of at least three independent measurements.

Figure 2

LCPUFAs uniquely alter the aggregation rate of insulin. ThT aggregation kinetics (a) with a histogram (b) that summarizes the mean values (±SEM) of t_lag, t_1/2, and t_grow of insulin in the lipid-free environment (gray), as well as in the presence of C20:3 (yellow), C20:5 (orange), and C22:6 (red). P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001. “NS” nonsignificant difference. Each kinetic curve is the average of at least three independent measurements.

Figure 3

LCPUFAs uniquely alter the aggregation rate of insulin. ThT aggregation kinetics (a) with a histogram (b) that summarizes the mean values (±SEM) of t_lag, t_1/2, and t_grow of insulin in the lipid-free environment (gray), as well as in the presence of C18:2 (yellow), C18:3 (orange), C18:4 (red), and C18:3 (green). P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001. “NS” nonsignificant difference. Each kinetic curve is the average of at least three independent measurements.

Figure 4

Morphology of insulin aggregates grown in a lipid-free environment (Ins) and in the presence of LCUFAs and LCPUFAs. White scale bars are 500 nm. Z scale bars are in nm.

Figure 5

Insulin aggregates grown in the presence of LCUFAs and LCPUFAs exert different cell toxicity compared to that of the aggregates grown in the LCPUFA-free environment. Histograms of LDH (a), ROS (b), and JC-1 (c) assays of insulin aggregates grown in the presence of LCUFAs and LCPUFAs, as well as in the lipid-free environment (Ins). Black asterisks (*) show a significance level of differences between protein aggregates and the control; red asterisks (*) show a significance level of differences between Ins and protein aggregates grown in the presence of LCUFAs and LCPUFAs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. “NS” nonsignificant difference.