Insights into Insulin Fibril Assembly at Physiological and Acidic pH and Related Amyloid Intrinsic Fluorescence.

Clara Iannuzzi,Gaetano Irace,Ivana Sirangelo,Marianna Portaccio,Margherita Borriello

doi:10.3390/ijms18122551

Clara Iannuzzi, Gaetano Irace + Show 3 more

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https://doi.org/10.3390/ijms18122551

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Abstract

Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property.

Highlights

Insulin is a small helical protein and a key hormone regulating glucose homeostasis
We have investigated the amyloid fibril formation in insulin under these conditions using both Thioflavin T (ThT) fluorescence assay and transmission electron miscroscopy (TEM) imaging
These results suggest that fibril formation in insulin at neutral and acidic pH might proceed through different pathways leading to structurally different amyloid fibrils

Summary

Introduction

Insulin is a small helical protein and a key hormone regulating glucose homeostasis. This protein is made up by two peptide chains, the A and B chain, linked by two disulfide bonds. Insulin has been shown to aggregate forming amyloid fibrils in the site of medication injections of diabetic patients causing a pathological condition, named insulin injection amyloidosis [10,11,12,13,14,15]. In this pathology, insulin amyloid fibrils form a hard subcutaneous mass at the injection site and an immune response may be triggered. The insulin fibril formation causes serious therapeutic problems such as poor glycemic control because of the impairment in insulin absorption, and catheter occlusions during continuous subcutaneous insulin infusion [16,17]

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