Oleate rescues INS-1E β-cells from palmitate-induced ER-stress through a reduced phosphorylation of eIF2α

Aims: Free fatty acids (FFAs) are elevated in hyperlipidaemia which is a common pathology in obese individuals and often associated with diabetes. Therefore, FFAs, whose secretion from adipose tissue is increased in obesity, have been suggested to link obesity and type 2 diabetes. Saturated FFAs, such as palmitate, cause β-cell apoptosis which could be mediated through persistent endoplasmic reticulum (ER)-stress which is also called Unfolded Protein Response (UPR).

We questioned if palmitate induces β-cell apoptosis through an activation of the ER-stress signalling pathway Unfolded Protein Response and whether oleate as a monounsaturated fatty acid can counteract these effects?

Methods: INS-1E β-cells were incubated with palmitate, oleate or the combination of both. Viability was measured using WST-1 assay. Apoptosis was determined by AnnexinV/Propidium-Iodide-Assay. For further characterization of the UPR, the activation of the three main pathways, ATF6, PERK and IRE1was analyzed by Western blotting.

Results: Palmitate significantly decreased viability (29 ± 8.8%) of INS-1E cells compared to cells treated with serum-free medium (SFM) after 24h incubation. In contrast, viability was not changed after 6h incubation with palmitate. Stimulation with oleate showed no effect on viability but the combination of oleate and palmitate improved viability compared to palmitate treated cells (55 ± 9.3%) or controls (26 ± 5.3%). The number of apoptotic cells was increased 2-fold after 24h incubation with palmitate compared to control cells. Again, oleate alone showed no effect but in combination ameliorated the effect of palmitate to control level. Apoptosis was not detected after 6h incubation with palmitate.

Phosphorylation of eIF2α was increased after 6 and 24h incubation with palmitate demonstrating an activation of the PERK pathway. In contrast, oleate had no effect. Furthermore, no activation was detected after stimulation with the combination of palmitate and oleate. eIF2α protein levels were also increased by palmitate after 6 and 24h, but not by oleate. The combination of palmitate and oleate counteracted the effects of palmitate. Protein levels of BiP, a target of the IRE1 and ATF6 pathway, were not influenced by palmitate or oleate.

Conclusion and outlook: Palmitate as a saturated fatty acid induced lipotoxicity in INS-1E cells by prolonged ER-stress. Oleate as a monounsaturated fatty acid had no toxic effects on β-cells but could ameliorate the effects caused by palmitate. For better analysis of the palmitate-induced ER-stress, levels of chaperones will be measured using quantitative real time PCR. To show probable changes in the lipid composition of cell- and ER-membrane, MALDI-TOF analysis will be performed.