Excessive Iron Induces Oxidative Stress Promoting Cellular Perturbations and Insulin Secretory Dysfunction in MIN6 Beta Cells.

Hisham Al-Obaidi,Derek Renshaw,Vinood B. Patel,Mohammed Gulrez Zariwala,Voni Blesia

doi:10.3390/cells10051141

Hisham Al-Obaidi, Derek Renshaw + Show 3 more

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https://doi.org/10.3390/cells10051141

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Abstract

Exposure to high levels of glucose and iron are co-related to reactive oxygen species (ROS) generation and dysregulation of insulin synthesis and secretion, although the precise mechanisms are not well clarified. The focus of this study was to examine the consequences of exposure to high iron levels on MIN6 β-cells. MIN6 pseudoislets were exposed to 20 µM (control) or 100 µM (high) iron at predefined glucose levels (5.5 mM and 11 mM) at various time points (3, 24, 48, and 72 h). Total iron content was estimated by a colourimetric FerroZine™ assay in presence or absence of transferrin-bound iron. Cell viability was assessed by a resazurin dye-based assay, and ROS-mediated cellular oxidative stress was assessed by estimating malondialdehyde levels. β-cell iron absorption was determined by a ferritin immunoassay. Cellular insulin release and content was measured by an insulin immunoassay. Expression of SNAP-25, a key protein in the core SNARE complex that modulates vesicle exocytosis, was measured by immunoblotting. Our results demonstrate that exposure to high iron levels resulted in a 15-fold (48 h) and 4-fold (72 h) increase in cellular iron accumulation. These observations were consistent with data from oxidative stress analysis which demonstrated 2.7-fold higher levels of lipid peroxidation. Furthermore, exposure to supraphysiological (11 mM) levels of glucose and high iron (100 µM) at 72 h exerted the most detrimental effect on the MIN6 β-cell viability. The effect of high iron exposure on total cellular iron content was identical in the presence or absence of transferrin. High iron exposure (100 µM) resulted in a decrease of MIN6 insulin secretion (64% reduction) as well as cellular insulin content (10% reduction). Finally, a significant reduction in MIN6 β-cell SNAP-25 protein expression was evident at 48 h upon exposure to 100 µM iron. Our data suggest that exposure to high iron and glucose concentrations results in cellular oxidative damage and may initiate insulin secretory dysfunction in pancreatic β-cells by modulation of the exocytotic machinery.

Highlights

Synaptosomal associated protein 25 (SNAP-25) binds to syntaxin 1 and VAMP2 and together the three SNARE proteins generate a steady bundle of the four α-helices, with each protein containing one α-helix, and two α-helices from SNAP23/25 [4]
The results show insulin secretion levels following treatment with glucose compared to control (Figure 7a), iron against control (Figure 7b), combinations of iron and glucose against control (Figure 7c), and measurements of iron, glucose, and combinations of iron and glucose at 5, 10, 30, and 60 min (Figure 7d), which was applied at 24 h incubation (Figure 7e)
After 24 h of incubation, it decreased the SNAP-25 levels significantly compared to that in the control (86.61 ± 2.71 vs. 293 ± 0.42). These results suggest that 100 μM iron and 11 mM glucose concentrations significantly reduce the levels of SNAP-25 in MIN6 cells even after 24 h

Summary

Introduction

Insulin is a key homeostatic regulator of blood glucose Defects in both insulin secretion and insulin action contribute to the development of T2DM [3]. SNAP-25 binds to syntaxin 1 and VAMP2 and together the three SNARE proteins generate a steady bundle of the four α-helices, with each protein containing one α-helix (from VAMP and syntaxin), and two α-helices from SNAP23/25 [4] These proteins have a crucial role in regulating = vesicle/granule exocytosis of insulin, and sharing numerous commonalities with neuronal synaptic vesicle exocytosis [4]. Reactive oxygen species (ROS) provide beneficial effects in many metabolic processes [7] These powerful agents can be important mediators of damage to cell structures, nucleic acids, lipids, and proteins, that is associated with biochemical and functional changes [8]. Lipid peroxidation is one biological process resulting from the attack by metalinduced generation of ROS, producing malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) [9]

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