ER stress and activation of UPR has been suggested as a mechanism of how raised nutrient levels exert their detrimental effects on palmitate-treated beta-cells. Support for a role of ER stress in lipotoxicity via activation of the UPR has been supplied by studies, where manifestations of the UPR were recorded in beta-cells undergoing apoptosis as a consequence of exposure to palmitate [4–7, 9, 10]. In triggering ER stress saturated fatty acids such as palmiate are much more potent than unsaturated fatty acids, such as oleate . In line with this, knocking-down SCD1 in palmitate-treated beta-cells aggravates the toxic effect of the saturated fatty acid .
The mechanisms of how fatty acids induce ER stress are yet unclear. Some authors hypothesize that prolonged exposure to palmitate causes ER Ca2+ release, which disturbs ER homeostasis and causes ER stress [24, 34]. However, it is unlikely that the observed modest depletion of ER Ca2+ may lead to ER stress . Another argument against this hypothesis is that release of ER Ca2+ was observed in cells treated with oleate, which does not induce ER stress [24, 34]. ER protein overload is another mechanism, which may contribute to ER stress in lipotoxic beta-cells. Recent studies showed that protein overload might be a consequence of reduced ER-to-Golgi protein trafficking or degradation of carboxypeptidase E [35, 36]. However, absence of significant changes in the expression level of molecular chaperones, such as BiP, in the current and previous studies makes this hypothesis questionable [4, 10, 11]. Up-regulation of ER chaperones is a major response to accumulation of unfolded protein in the ER. The protective role of BiP is also questioned in the study, where over-expression of BiP in INS-1 cells and MIN6 cells partially reduced susceptibility to thapsigargin but failed to reduce palmitate-induced ER stress . Furthermore, gene expression analysis did not detect changes in mRNA level of ER chaperones in palmitate-treated beta-cells . It should also be mentioned that in some studies results concerning expression and role of BiP in palmitate-treated beta-cells are opposite. Thus, Kharroubi et al. demonstrated palmitate-induced up-regulation of BiP mRNA level connected with enhanced apoptosis . Also, Laybutt et al. observed protective, ER stress-reducing and anti-apoptotic effects of BiP over-expression .
Prolonged exposure to palmitate leads to formation of tripalmitin . It was shown that tripalmitin accumulates in the ER rather than in the cytoplasm of the beta-cells. This is in contrast to oleate, which forms droplets in the cytoplasm . Accumulation of insoluble tripalmitin may cause morphological perturbations in the ER and be toxic for the beta-cells [17, 18]. We hypothesized that reducing fatty acid oxidation would accentuate ER stress by increasing generation and incorporation of tripalmitin into microsomes. To reduce metabolism we raised glucose, which inhibits palmitate oxidation and shuttles the fatty acid to non-oxidative pathways [20, 23]. Also, we used AMPK activator AICAR that stimulates, and CPT1 inhibitor etomoxir that inhibits oxidation [19, 22, 23]. Fatty acid oxidation was higher at low glucose compared to high glucose concentration both in cell lines and human islets, but especially pronounced in INS-1E cells. Accentuated glucose-induced reduction in palmitate oxidation in INS cells was also observed in previous studies [23, 38]. More efficient oxidation of fatty acids by MIN6 cells and human islets compared to INS-1E cells in the presence of high glucose might be explained by higher activity of ACC in INS-1E cells . The accentuated glucose-induced reduction in palmitate oxidation in INS-1E cells may explain the significant effect of AICAR in these cells. Surprisingly, essentially similar activation of UPR by palmitate was observed both at low and high glucose concentrations. Furthermore, stimulation and inhibition of palmitate oxidation by AICAR and etomoxir did not affect palmitate-induced UPR activation. This holds true for both cell lines and human islets. More robust activation of UPR markers in INS-1E cells may speak in favor of higher sensitivity of these cells to palmitate exposure.
Previous data on the role of glucose and fatty acid metabolism in palmitate-induced ER stress response are controversial. While Bachar et al claimed that glucose amplified ER stress , Cunha et al showed that glucose did not amplify ER stress in palmitate-treated beta-cells . The discrepancy was suggested to be explained by the fact that protein and mRNA levels of ER stress markers were analyzed by the respective groups. Inhibition of CPT1 by siRNA approach performed by Choi et al caused increased expression of CHOP but decreased phosphorylation of eIF2α and JNK .