Palmitate is selectively toxic to rodent but not human β-cells. Palmitate (C16:0) is the most abundant LC-SFA in the circulation , and the effects of chronic exposure (≥ 24 h) of rodent INS-1E and human EndoC-βH1 cells to palmitate were investigated. As expected, C16:0 caused a dose-dependent loss of viability when INS-1E cells were treated with increasing concentrations of C16:0 for 24 h (Fig. 1a). Surprisingly however, over this time course, exposure to C16:0 did not cause any loss in viability of EndoC-βH1 cells. This was also true even when cells were exposed to this fatty acid at concentrations as high as 500 µM, over an extended exposure period of up to 72 h (Fig. 1b). Similarly, C16:0 [500µM] was well tolerated by EndoC-βH1 cells even in the presence of high glucose [20mM] (Fig. 1c) which has previously been suggested to exacerbate lipotoxicity in rodent β-cells .
In one recent study , similar results were obtained but it was proposed that the response varied according to the culture medium in which the cells were grown. Therefore, we substituted the standard growth medium for EndoC-βH1 cells, DMEM, with a second medium, DMEM/Ham’s F12 mixture, since the latter has been proposed to render the cells sensitive to the toxic effects of C16:0 . However, we observed no increase in EndoC-βH1 cell death when cells were treated with C16:0 [500µM] in DMEM/Ham’s F12 medium (Fig. 1d).
Effects of other long-chain saturated fatty acids (LC-SFA) on the viability of rodent and human β-cells. Next, we sought to explore if fatty acid chain length was a determinant of the extent of toxicity in rat or human-derived β-cells. Exposure of INS-1E cells to 500µM of the LC-SFAs, pentadecanoic (C15:0), heptadecanoic (C17:0), stearic (C18:0) and nonadecanoic (C19:0) acid, resulted in a dramatic increase in cell death after 24 h (Fig. 2a). However, toxicity did not correlate directly with chain length, since, when INS-1E cells were incubated with FFAs at the very high concentration of 500µM, C15:0 caused significantly (p = 0.002) greater cell death than was seen with longer chain saturated species. This was also true when a lower concentration [250µM] was employed (as shown in Additional file 1: Supplementary Fig. 1).
By contrast with rodent β-cells, human EndoC-βH1 cells were much less sensitive to the detrimental effects of 500 µM of long chain FFAs such as C15:0, C17:0 or C18:0 (Fig. 2b) since their viability was maintained at high levels even after 72 h of treatment. Strikingly, exposure of the EndoC-βH1 cells to C19:0 caused a marked increase (p < 0.0001) in death (Fig. 2b) although, even in this case, the lipotoxic effects were less potent than in INS-1E cells (compare Fig. 2c + d). The loss of viability of EndoC-βH1 cells required concentrations of ≥ 500 µM whereas, in INS-1E cells, death was increased significantly at concentrations as low as 125 µM.
Long-chain monounsaturated fatty acids are well tolerated by both human and rodent β-cells. In confirmation of previous studies [7, 24], the long-chain monounsaturated fatty acid (LC-MUFA), oleic acid (C18:1; 250µM), did not promote any loss of viability in INS-1E cells. Moreover, co-incubation of these cells with C16:0 and C18:1 for 24 h led to an attenuation of the extent of cell death seen with C16:0 alone (Fig. 3a). Similar, to the situation in INS-1E cells, exposure of EndoC-βH1 cells to C18:1 was not associated with any loss of viability (Fig. 3b).
LC-FFAs attenuate cytokine induced β-cell death in EndoC-βH1 cells. We, and others, have previously shown that culture in the presence of a cocktail of pro-inflammatory cytokines induces the loss of viability in rodent β-cells. Moreover, we also discovered that this response was inhibited upon inclusion of the LC-MUFAs, C16:1 or C18:1, in the incubation medium . In the present work, we have confirmed that EndoC-βH1 cells also succumb to pro-inflammatory cytokines (Fig. 4), confirming previous reports by ourselves [7, 12] and others . Surprisingly, however, we found that, when 250µM of C16:0, C16:1 or C18:1 were also present in the incubation medium, cytokine-induced cell death was attenuated significantly (although, LC-FFAs did not offer complete protection against cytokine induced death under these conditions).