supplied by diet or circulating unbound free fatty acids in the aqueous phase, has been suggested to activate the fatty acid G- protein coupled receptor (GPR40). Activation of GPR40 causes an increase in intracellular Ca2+, which is thought to be induced via activation of the Gaq-phospholipase C pathway. The increase in free cytosolic Ca2+ plays a crucial role in stimulating insulin secretion [14]. Although important for beta cell function, sustained increases in fatty acid availability and influx, as occur in response to high fat feeding [6], can induce both dysfunction [7] and death of beta cells [16] and thereby contribute to the pathogenesis of diabetes mellitus. Sustained exposure of pancreatic beta cells to fatty acids, such as palmitate, has been linked to a loss in GSIS and increased apoptosis [7,17–20]. What is less well understood is the mechanism underpinning the lipotoxic effects of palmitate. Analysis of mice lacking GPR40 indicate that although important in mounting acute responses to fatty acid supply the receptor is unlikely to contribute to pancreatic dysfunction induced in response to sustained increases in fatty acid availability [14]. While there is no compelling evidence implicating other fatty acid receptors or transporters an increase in fatty acid provision has been suggested to disrupt the glucose-fatty acid cycle by inhibition of pyruvate dehydrogenase, which switches fuel consumption from glucose to fatty acid oxidation [18,21]. Fatty acids may also induce much greater oxidation by increasing the expression of CPT-1 [22] whilst decreasing that of acetyl CoA carboxylase [23]. This exaggerated fatty acid oxidation may promote beta cell insensi- tivity to glucose resulting in a concomitant reduction in GSIS, which may also be underpinned by defects in the regulation or expression of transcription factors, such as PDX-1 [24], or indeed that of the insulin gene itself [25]. In many cell types increased provision of fatty acids has been linked to enhanced intracellular synthesis and accumulation of ceramide, a sphingolipid that can be generated de novo from saturated fatty acids (e.g. palmitate) via the seine palmitoyl transferase (SPT) pathway. Ceramide has been implicated strongly in cell apoptosis and in the pathogenesis of insulin resistance and there is evidence in the literature suggesting that it may be an effector molecule mediating palmitate-induced beta cell dysfunction [26,27] and beta cell death [6]. Here we show that sustained exposure of pancreatic MIN6 beta cells to palmitate results in a substantial loss in insulin secretion in response to glucose or amino acid provision. This fatty acid- induced impairment in insulin secretion blunts autocrine signalling by the hormone, but, unlike in other cell types, has no detectable effect on PKB-directed insulin signalling in response to exoge- nously supplied hormone. Furthermore, we demonstrate that the palmitate-induced reduction in insulin secretion cannot be attributed to ceramide action, but may be due, in part, to a loss in nutrient-induced CaMKII and ERK signalling which normally supports the stimulus-dependent secretion of insulin. Results Palmitate induces a loss in glucose- and amino acid- stimulated insulin secretion and induces apoptosis in MIN6 b cells Insulin release from pancreatic b cells is normally negligible in the presence of fasting blood glucose levels (i.e. ,3–4 mM). However, in response to an increase in extracellular glucose (as occurs during the post absorptive phase when it may rise by 2–4 fold, i.e. 7–16 mM) or indeed in response to increased amino acid provision, secretion of insulin is enhanced significantly [28]. This glucose- and amino acid-dependent response is illustrated in Figure 1A, which shows a significant increase (up to 7.5-fold) in Palmitate and Beta Cell Insulin Secretion insulin secretion when MIN6 b cells were incubated in media containing 16 mM glucose or a 3 mM glucose/10 mM leucine and glutamine mix (shown previously to stimulate insulin secretion independently of glucose metabolism [29]). Irrespective of the stimulus used, this nutrient-dependent increase in insulin secretion was significantly blunted when MIN6 b cells were preincubated with palmitate (0.4 mM) for a period of 48 h (Figure 1A) falling by ,75% when expressed as a net change (Figure 1B). Intriguingly, even in the absence of a nutrient stimulus we observed a significant reduction in basal insulin secretion indicating that palmitate over supply may also target the secretory process that operates in response to a sub-threshold glucose (3 mM) stimulus. Given that palmitate is known to induce apoptosis of b cells it is plausible that the loss in GSIS may be related to a reduction in MIN6 cell number. However, analysis of adherent cell numbers based on DAPI staining did not reveal any significant changes in control or palmitate-treated cell populations (Figure 2A and 2B) and, in separate experiments, we could not detect any additional effect upon cell viability of subjecting palmitate-treated MIN6 cells to a glucose or amino acid stimulus suggesting that cells were able to tolerate the combind stress of palmitate exposure and energy stimulation (Figure 2C). Nonetheless, it is noteworthy that palmitate treatment induces a 2-fold increase in caspase 3/7 activity (a proapoptotic marker) in MIN6 cells (Figure 2B), suggesting that whilst apoptosis had been initiated by the fatty acid this had not yet translated to a reduction in cell number over the 48 h incubation period being studied. However, it is noteworthy that in preliminary experiments analysis of cell number following 60 h of treatment with palmitate had revealed a significant decline in cell survival; an observation that we had used to help to inform our choice of using a shorter palmitate incubation period for the studies reported in this paper (data not shown). The loss in GSIS cannot be attributed to ceramide action Palmitate-driven ceramide synthesis has been shown to impair insulin action and induce apoptosis in a number of different cell types and evidence exists in the literature implicating ceramide in b-cell dysfunction. To test whether ceramide may be an effector molecule mediating the lipid-induced loss in GSIS in MIN6 b cells we investigated the effect of two inhibitors that suppress de novo ceramide synthesis, AZ422 and fenretinide. AZ422 inhibits the activity of SPT (Figure S1) and fenretinide suppresses the conversion of dihydroceramide to ceramide by inhibiting dihy- droceramide desaturase. Both inhibitors failed to antagonise the palmitate-induced loss in GSIS indicating that any increase in ceramide synthesis was unlikely to account for the impaired secretion of insulin in response to palmitate (Figure 3A). Consistent with this proposition, raising cell ceramide content by incubating MIN6 b cells with increasing concentrations of C2-ceramide (a short-chain cell permeant ceramide analogue) did not alter GSIS when compared to output of insulin from untreated cells or those incubated with the inert C2-dihydroceramide analogue (Figure 3B). Ceramide concentrations above 50mM were not used as these proved cytotoxic. It is important to stress that whilst 50mM C2-ceramide did not affect GSIS this concentration of ceramide was effective in promoting phosphorylation/activation of atypical PKCs (a known ceramide target), whereas the inactive C2- dihydroceramide did not (Figure 3C). In both adipocytes and muscle cells ceramide is a potent inhibitor of PKB/Akt activation [30,31]. However, it is noteworthy that in MIN6 b cells activation of PKB in response to GSIS was unaffected by cell incubation with C2-ceramide (Figure 3D). Analysis of cell ceramide content following a 48 h palmitate incubation unexpectedly revealed that PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25975