Insulin synthesis increases the levels of hydrogen peroxide in the lumen of the endoplasmic reticulum and induces UPR

Background and aims:

Early stages of type 2 diabetes (T2DM) are characterized by high requirements of insulin to compensate insulin resistance. Increasing insulin synthesis and folding has been proposed as a cause of H2O2 generation, unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. Combined with the low expression of antioxidative enzymes in β-cells, this could cause β-cell dysfunction. However, confirming H2O2 production in the ER lumen has been limited by its thiol-oxidizing environment.

Methods:

RINm5F cells were transfected with TriPer, a novel fluorescent protein capable to sense H2O2 in the ER. To study the formation of H2O2 secondary to increased insulin biosynthesis in the ER, and to exclude side effects of glucose-metabolism, RINm5F cells were co-transfected with a doxycycline-inducible expression system, either for wild type human insulin (Tet-On-WT-hINS) or for misfolded insulin (Tet-On-C96Y-hINS). After doxycycline administration, TriPer response was analyzed by microscopy, UPR gene expression by qPCR and cell viability by MTT-assay.

Results:

Reduced fluorescence ratios of TriPer (488/405nm) were detected in both models after insulin induction, confirming the production of H2O2 as result of insulin synthesis. Higher concentrations of H2O2 were detected after C96Y-hINS expression, equivalent to 100µM exogenously administrated H2O2. UPR was activated, however, only C96Y-hINS expression triggered apoptotic pathways reducing cell viability.

Conclusions:

Insulin biosynthesis increases H2O2 inside the ER without causing apoptotic signals. However, accumulation of unfolded/misfolded insulin activates apoptosis and cell death. A long course of high insulin demand could lead to misfolded insulin accumulation, H2O2 generation and β-cell dysfunction in T2DM.