Palmitate and Beta Cell Insulin Secretion Figure 2. Effects of palmitate on MIN6 b cell number and apoptosis. MIN6 b cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h prior to analysis of cell number by DAPI staining (A) or (B) caspase 3/7 activity (a proapoptotic marker). Three random visual fields from duplicate samples were chosen (typical phase, DAPI stained and merged images are shown in panel A), and adherent cell number noted from control or palmitate-treated cells following DAPI staining (A and B). Alternatively, following the fatty acid incubation period cells were incubated with caspase 3/7 assay reagent and caspase 3/7 activity determined by luminescence as described in methods (B). In some experiments, cells that had been pre-treated with palmitate (0.4 mM, 48 h) were subsequently incubated with glucose (16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h prior to analysis of cell number by DAPI staining (C). Bar values are mean 6 SEM from 3 or 4 separate experiments. The asterisk signifies a significant difference from the appropriate control value (P,0.05). doi:10.1371/journal.pone.0025975.g002 Impaired GSIS and ERK1/2 signalling in MIN6 b cells is not dependent upon fatty acid oxidation To assess whether the suppressive effect of palmitate upon ERK signalling and GSIS involves mitochondrial fatty acid metabolism we investigated the effect of 2-bromopalmitate, a non-oxidisable analogue of palmitate. Figure 7A shows that 2-bromopalmitate proved just as effective as palmitate in suppressing GSIS and, despite its lack of metabolism, was just as effective in suppressing activation of ERK, PKB and CaMKII (Figure 7B). To further substantiate that fatty acid oxidation was unlikely to be an important determinant of the palmitate-induced loss in GSIS, MIN6 b cells were incubated with either carnitine or etomoxir. PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25975