Palmitate and Beta Cell Insulin Secretion Figure 1. Palmitate induces a loss in glucose- and amino acid-stimulated insulin secretion. (A) MIN6 b cells were incubated with palmitate (0.4 mM) for 48 h prior to incubation with glucose (3 mM or 16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h. Glucose-stimulated insulin secretion (GSIS) and amino acid-stimulated insulin secretion (AASIS) were then assessed as described in the methods. The net change in insulin secretion from 3 to 16 mM glucose or to 10 mM amino acid was analysed and graphed (B). Values are mean 6 SEM from 3 separate experiments. The asterisk signifies a significant difference from the appropriate control (P,0.05). doi:10.1371/journal.pone.0025975.g001 there was little, if any, detectable ceramide synthesis from palmitate in MIN6 b cells (Figure 3E). The DAG kinase assay that we employed readily detected significant increases in cell ceramide following cell treatment with C2-ceramide (Figure 3E) and in palmitate-driven DAG synthesis (Figure 3F). Incubation with C2-dihyrdoceramide did not register any increase in ceramide consistent with the fact that it does not serve as a substrate for DAG kinase (Figure 3E). Effects of palmitate on autocrine insulin signalling in MIN6 b cells The effect of palmitate on insulin signalling was subsequently investigated in MIN6 b cells. Under conditions of low (3 mM) glucose the phosphorylation of PKB and that of ERK1 and ERK2 was not readily detectable, but was enhanced significantly upon incubation of MIN6 b cells with high (16 mM) glucose (Figure 4A and 4B). This glucose-induced increase in PKB and ERK phosphorylation was blunted significantly upon preincubation of MIN6 b cells with palmitate (Figure 4A and 4B). To assess whether this palmitate- induced loss in PKB and ERK phosphorylation was due to impaired GSIS, insulin was added exogenously to the media bathing MIN6 b cells in the absence and presence of palmitate. This experimental approach revealed that palmitate was unable to blunt the activation of PKB in response to exogenously added insulin (Figure 4A) and that the impaired activation of PKB seen in response to high glucose was more likely explained by the inhibitory effect of palmitate on insulin secretion rather than insulin signalling per se. In striking contrast, exogenously supplied insulin did not activate ERK and was unable to over-ride the inhibitory effect of palmitate on glucose-stimulated ERK phosphory- lation (Figure 4B). This latter observation clearly implies that glucose- induced ERK activation is not reliant upon autocrine activation of proximal insulin-signalling components and depends upon a mecha- nism that is distinct from that promoting activation of PKB. Activation of ERK and CaMKII is required to support glucose- and amino acid-induced insulin secretion The finding that palmitate blunts glucose-stimulated ERK signalling and also induces an attendant reduction in insulin secretion implies that activation of ERK may be required to support stimulus-induced insulin secretion. To test this proposition we assessed the effect of U0126, a selective inhibitor of the MEK- ERK signalling cascade, upon both glucose- and amino acid- induced ERK activation and insulin secretion. Figure 5A shows that increased provision of glucose or amino acids induces activation of ERK signalling, which was suppressed by coincuba- tion of cells with U0126. Associated with this inhibition in ERK activation by U0126 was a significant reduction (by up to 62%) in insulin secretion from MIN6 b cells in response to increased availability of both glucose and amino acids (Figure 5B). Previous work has indicated that glucose-induced increases in cytosolic calcium can activate the ERK pathway via stimulation of CaMKII and that this activation occurs independently of Ras and Raf [32]. We therefore assessed the effects of CaMKII inhibition on both glucose-induced ERK activation and insulin secretion. Figure 6A and 6B shows that KN-62, a CaMKII inhibitor, antagonised both ERK activation and insulin secretion in response to increased glucose and amino acid provision. The notion that CaMKII may serve as an intermediary in the activation of ERK signalling and GSIS is further strengthened by the observation that phosphorylation/activation of CaMKII itself in response to glucose and amino acids was reduced following palmitate treatment (Figure 6C). PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25975