Increased toxicity of amylin (Islet Amyloid Polypeptide) in beta cells induced by photochemical internalization

Amylin and oligomers formed from amylin are implicated in demise of beta cells in type 2 diabetes. However, whether putative toxicity is exerted intra or extracellularly is unclear. Use of photochemical internalization (PCI) technique may give clues for impact of intracellular toxicity.

(a) To optimize the concentration and exposure set up of the photosensitizing compound meso-disulfonated tetraphenyl chlorin TPCS_2a (Amphinex^®) for use in insulin producing beta cells and (b) to utilize the photosensitizing technique to probe for intracellular effects in beta cells by amylin.

The titration of TPCS_2a and blue light exposure was evaluated by MTT assay. The insulin producing INS-1 832/13 beta cells were incubated with the photosensitizing agent TPCS_2a prior to exposure of amylin. Viability and function were further evaluated by standard biochemical techniques.

A protocol was developed for use in INS-1 832/13 cells in which the optimal concentration of TPCS_2a was found to be 4ng/ml. Using this protocol human amylin (10 μM, 8 h) in combination with TPCS_2a (4 ng/ml, 18 h) and blue light exposure (60 s) exerted toxic effects above those by TPCS_2a and illumination alone as measured by MTT (15 ± 3.6%, n = 6, p < 0.007) for effect of amylin exposure. On the other hand, rat amylin (which does not form oligomers) had no effect. Insulin secretion was non-significantly reduced by the combination of human amylin with TPCS_2a and illumination compared to TPCS_2a and illumination alone. Cellular insulin content was not affected, nor were measured parameters of apoptosis and necrosis.

PCI technology could be a useful tool to induce endosomal rupture in clonal beta cells. The present results using PCI are compatible with intracellular negative effects following exposure to amylin.