(308b) Antioxidative Oligomeric Procyanidins Prevent Insulin Fibrillation Via the Formation of Unstructured, off-Pathway Aggregates

β-sheet–rich amyloid fibrils or aggregates accumulation has been implicated in a number of human diseases [Ross and Poirier, 2004; Stoppini et al, 2004]. Numerous studies demonstrate that natural polyphenols decrease the risk of degenerative diseases and inhibit in vitro amyloid formation. However, the molecular mechanism for the anti-amyloidogenesis of polyphenols is still unclear. We have investigated the effects of oligomeric procyanidins (OPCs), resveratrol, and trehalose on the amyloidogenicity of insulin using thioflavin-T fluorescence, dynamic light scattering, circular dichrosim, transmission electronic microscopy, and cyclic voltammograms. The main ideas and conclusions are summarized as follows.

(1) The order of inhibitory effect was OPCs > resveratrol > trehalose, suggesting that polyphenolic structure is essential for fibril deposition, as observed by ThT fluorescence. DLS results showed that the presence of OPCs significantly inhibited the aggregation process, at which only smaller particles (d_H = 15 nm and 200 nm) were found after incubation for 105 h. Meanwhile, resveratrol and trehalose failed to show an apparent inhibitory effect on protofibrils, with large particles observed at a similar size to that of insulin alone. TEM results indicated that OPCs show potent inhibitory effects at all stages of insulin fibrillation and redirect insulin aggregation pathway via the formation of unstructured, off-pathway aggregates.

(2) Electrochemical measurements indicate that anti-amyloidogenic effect also correlates with the antioxidative activity of amyloid inhibitor. OPCs were found to be more reducing than resveratrol and trehalose, with evident redox peaks at 0.441 and 0.385 V, respectively. The potential difference (ΔE) between the anodic and cathodic peaks was approximately 56 mV. The anodic peak current was nearly equal to the cathodic peak current, indicating a reversible process that involves one proton and one electron [Liu R et al, 2011]. Meanwhile, the OPCs solution showed a gradual decrease in current intensity (E = −0.624 V) with the increase in incubation time, and the reduction peak potential slightly shifted from −0.624 to −0.61V; whereas in the resveratrol and trehalose solutions, only shifts in the reduction peak potential were observed. These results suggest that the antioxidant capabilities of the compounds weakened with increased incubation time.

(3) This study showed, for the first time, how the aggregation pathway of bovine insulin can be redirected with OPCs. The interactions among aggregation-prone domains of bovine insulin were suppressed via the formation of OPC-containing, unstructured off-pathway aggregates, whereas this phenomenon was not observed in the presence of resveratrol, suggesting that the properties of proteins, such as hydrophobicity and charge, play a crucial role in regulating the aggregation pathway, and that OPCs are more accessible than resveratrol for insulin intermolecular interactions.

In summary, the present work demonstrated that OPCs in grape seeds have potent inhibitory and disruptive effects on amyloid fibrillar structures in vitro, inducing the transformation of insulin monomers into unstructured, off-pathway aggregates. The structural properties of compounds, including aromatic structures, and the number of hydroxyl groups must be taken into account in disrupting the aggregation pathway of proteins. Electrochemical measurements indicated that the anti-amyloidogenic effect was also correlated with the antioxidative activity of the amyloid inhibitor. These findings contribute to the development of novel anti-amyloidogenic products from naturally occurring materials.

This work was supported by the Natural Science Foundation of China (No. 20806057 and 31071509), the Ministry of Science and Technology of China (Nos. 2012BAD29B05 and 2012AA06A303), and the Ministry of Education (No. NCET-11-0372).

References

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