Characterization of Novel Insulin Fibrils That Show Strong Cytotoxicity Under Physiological pH

Yoshihara, Hiromu; Saito, Jun; Tanabe, Ayaka; Amada, Takuma; Asakura, Takeshi; Kitagawa, Kouki; Asada, Shinichi

doi:10.1016/j.xphs.2016.01.025

Cited by 33 publications

(33 citation statements)

References 54 publications

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“…Despite a clear relevance to diabetes, insulin assembly under physiological conditions is not well understood. A relatively small number of studies examined insulin aggregation at neutral pH and of these, only two explored insulin concentrations ≤100 μM . Here, we examined insulin aggregation pathways from soluble oligomeric to fully aggregated fibrillar assemblies in vitro at acidic and neutral conditions in the concentration range of 2.5–100 μM.…”

Section: Discussionmentioning

confidence: 99%

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

et al. 2017

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Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection-site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5-100 μM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross-linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 μM insulin. These findings suggest that oligomers may be on- and off-pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer-sized aggregates, which may be of physiological relevance and provides insight into injection-site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.

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Section: Discussionmentioning

confidence: 99%

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

et al. 2017

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“…Stabilization of transthyretin tetramers and insulin dimers in neutral pH inhibit their amyloid formation. 75,76 Binding of small molecules to amyloidogenic SEVI precursors stabilized and inhibited amyloid fibrillation. EGCG was found to interact with SEVI precursors by forming a covalent bond between lysine side chains and EGCG ( Fig.…”

Section: Stabilization Of Sevi Precursors (Methods 7)mentioning

confidence: 99%

“…Maintaining of stable structures of amyloidogenic precursors which prohibits triggering of aggregation as the equilibrium shifts to monomers under the two‐state transition between monomers and amyloid fibrils. Stabilization of transthyretin tetramers and insulin dimers in neutral pH inhibit their amyloid formation . Binding of small molecules to amyloidogenic SEVI precursors stabilized and inhibited amyloid fibrillation.…”

Section: Inhibition Of Sevi‐enhanced Hiv Viral Infectionmentioning

confidence: 99%

Semen‐derived amyloidogenic peptides—Key players of HIV infection

Lee

Ramamoorthy

2018

Protein Science

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Misfolding and amyloid aggregation of intrinsically disordered proteins (IDPs) are implicated in a variety of diseases. Studies have shown that membrane plays important roles on the formation of intermediate structures of IDPs that can initiate (and/or speed-up) amyloid aggregation to form fibers. The process of amyloid aggregation also disrupts membrane to cause cell death in amyloid diseases like Alzheimer's disease and type-2 diabetes. On the other hand, recent studies reported the membrane fusion properties of amyloid fibers. Remarkably, amyloid-fibril formation by short peptide fragments of highly abundant prostatic acidic-phosphatase (PAP) in human semen and are capable of boosting the rate of HIV infection up to 400,000-fold during sexual contact. Unlike the least toxic fully matured fibers of most amyloid proteins, the semen-derived enhancer of virus infection (SEVI) amyloid-fibrils of PAP peptide fragments are highly potent in rendering the maximum rate of HIV infection. This unusual property of amyloid fibers has witnessed increasing number of studies on the biophysical aspects of fiber formation and fiber-membrane interactions. NMR studies have reported a highly disordered partial helical structure in a membrane environment for the intrinsically disordered PAP peptide that promotes the fusion of the viral membrane with that of host cells. The purpose of this review article is to unify and integrate biophysical and immunological research reported in the previous studies on SEVI. Specifically, amyloid aggregation, dramatic HIV infection enhancing properties, membrane fusion properties, high resolution NMR structure, and approaches to eliminate the enhancement of HIV infection of SEVI peptides are discussed.

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“…We have investigated insulin fibrillation under conditions closely resembling a physiological one (pH 7.4) [26], in contrast to the common experimental conditions of insulin fibrillation, such as low pH, extremely high temperature, high concentration of salts, and/or the presence of organic solvent [27,28]. The anti-aggregation properties of three native polyphenols, resveratrol (trans-3, 4, 5-trihydroxystilbene, RES), epigallocatechin gallate (EGCG) and curcumin (CUR), along with flavanone glycoside-hesperidin (HES), were tested for human insulin aggregation using Thioflavin T (Th-T) fluorescence.…”

Section: Resveratrol Inhibits Insulin Fibrillation Differently Comparmentioning

confidence: 99%

Resveratrol prevents amyloid fibrillation of insulin by arresting it in a bioactive oligomeric form

Das

Bhakta

et al. 2019

Preprint

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Insulin fibrillation is a limiting factor for its long-term storage because of considerably reduced bioavailable moieties. Deposition of fibrillated insulin can also cause subcutaneous insulin amyloidoma. Toxic phenolic compounds along with Zinc are used during commercial preparation of insulin to stabilize it in a hexameric form. Designed or repurposed natural small molecules with anti-amyloidogenic properties could thus be attractive agents for preventing insulin fibrillation. Natural polyphenolic compounds which have been shown to serve as anti-amyloid agents for proteins associated with neurodegenerative diseases are potential candidates for such function.In this study we have demonstrated that resveratrol, a natural polyphenol, can not only prevent insulin fibrillation but can also preserve insulin in a bioactive oligomeric form even at high temperature. While investigating the influence of some natural polyphenols on human insulin (hINS) in a condition inductive to amyloid fibrillation at physiological pH, we found attenuation, to different extents, of insulin fibril formation. However, visualization of polyphenol-treated hINS revealed that resveratrol in particular has the unique ability to arrest hINS before the onset of fibrillation growth in soluble oligomeric forms with discrete spherical morphology. Importantly, insulin treated with resveratrol retains its full biological activity in vivo and exerts no toxicity towards cell lines. Structural characterization of the major population of resveratrol-induced insulin oligomers by cryo-EM and single particle 3D reconstruction revealed its morphology that resembled crystal structure of insulin hexamer formulated with phenolic compounds. Thus, our study suggests that resveratrol can be an effective nontoxic substituent of phenolic compounds for insulin preservation.

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Characterization of Novel Insulin Fibrils That Show Strong Cytotoxicity Under Physiological pH

Cited by 33 publications

References 54 publications

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

Semen‐derived amyloidogenic peptides—Key players of HIV infection

Resveratrol prevents amyloid fibrillation of insulin by arresting it in a bioactive oligomeric form

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