Insulin–eukaryotic model membrane interaction: Mechanistic insight of insulin fibrillation and membrane disruption

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Abstract

Injection of exogenous insulin in the subcutaneous mass has been a proven therapy for type II diabetes. However, chronic administration of insulin often develops local amyloidosis at the injection site, pathologically known as “Insulin Ball”. This reduces the insulin bioavailability and exacerbates the disease pathology. Thus, the molecular interaction between insulin and the recipient's membrane surface plays a co-operative role in accelerating the amyloidosis. This interaction, however, is different from the molecular interaction of insulin with the native membranous environment of the pancreatic β-cells. The differential membrane mediated interaction that directly affects the aggregation kinetics of insulin remains elusive yet intriguing to understand the mechanism of pathological development. In this study we have characterized the interactions of insulin at different states with model eukaryotic membranes using high and low-resolution spectroscopic techniques in combination with microscopic investigation. Our results show that insulin amyloid intermediates are capable of interacting with model membranes with variable functional affinity towards the different compositions. Fluorescence correlation spectroscopy confirms the aggregation states of insulin in presence of the eukaryotic model membranes while solid-state NMR spectroscopy in conjugation with differential scanning calorimetry elucidates the molecular interaction of insulin intermediates with the lipid head groups along with the acyl chains. Additionally, dye leakage assays support the eukaryotic model membrane disruption by insulin intermediates, similar to hIAPP and Aβ40, as previously reported. Thus, the present study establishes the distinct mode of interactions of insulin amyloid with pancreatic β-cell and general mammalian cell mimicking membranes.

Abbreviations

ThT
thioflavin T
ANS
8-anilinonaphthalene-1-sulfonic acid
TMR
tetramethylrhodaminesuccinimidyl ester
6-CF
6-carboxyfluorescein
amyloid beta
hIAPP
human islet amyloid polypeptide
FCS
fluorescence correlation spectroscopy
AFM
atomic force microscopy
CD
circular dichroism
TEM
transmission electron microscopy
NMR
nuclear magnetic resonance
ssNMR
solid state NMR
TDDS
transdermal drug delivery systems

Keywords

Insulin amyloid
Membrane
FCS
ssNMR
DSC
TEM
AFM

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