Figure 4. Palmitate does not inhibit PKB activation in response to exogenously added insulin but does prevent glucose- induced activation of ERK. MIN6 b cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h. Glucose (16 mM) was added for 1 h and insulin (100 nM) in the penultimate 10 min of this incubation period. Cells were harvested and immunoblotted for phospho-PKBSer473 (A) and phospho-ERKThr202/Tyr204 (B) and normalised against total protein. Bars represent mean 6 SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P,0.05). doi:10.1371/journal.pone.0025975.g004 obesity can overload b cells, prompting cellular dysfunction as characterised by a reduction in GSIS [7] and increased susceptibility to lipoapoptosis [35]. An increase in long chain CoAs and palmitate-derived lipids such as ceramide and DAG has been demonstrated in b cells of diabetic rodent models [6]. Ceramide has been widely implicated as a pro-apoptotic agent and given that it can be generated from palmitate, a plausible hypothesis is that pancreatic b-cell dysfunction and apoptosis is mediated by palmitate-driven ceramide synthesis. It thus follows that suppressing cellular production of ceramide may potentially have beneficial consequences with regard b cell function. However, our analysis has revealed that whilst sustained provision of palmitate impairs GSIS from MIN6 b cells the fatty acid does not appear, under these circumstances, to drive an increase in ceramide synthesis. MIN6 b cells express both subunits of serine Palmitate and Beta Cell Insulin Secretion palmitoyl transferase (SPT) and whilst we find the enzyme is competent in generating ceramide from palmitate in vitro (see Figure S1) our observations indicate that ceramide is unlikely to be a key effector underpinning the loss in GSIS in response to palmitate in intact MIN6 b cells. The finding that inhibiting de novo ceramide synthesis via SPT or indeed incubating cells with a cell permeant ceramide analogue had no effect on GSIS would be consistent with this proposition. Moreover, it is noteworthy that our observations are fully in line with very recent studies in MIN6 cells in which sustained exposure to palmitate failed to induce increases in intracellular ceramide synthesis [36] or in which inhibition of de novo ceramide synthesis [37] did not attenuate the inhibitory effect of palmitate on insulin secretion. An important finding to emerge from our work is that, unlike adipocytes and skeletal muscle cells [30,31], palmitate does not impair insulin signalling in MIN6 b cells given that prior incubation of cells with palmitate had no detectable effect on activation of PKB signalling in response to exogenously added insulin. In both fat and muscle cells the loss in insulin-signalling capacity associated with palmitate supply is mechanistically attributed to ceramide generated from the fatty acid [30,31]. However, such a mechanism appears not to operate in MIN6 b cells based on the finding that C2-ceramide does not inhibit PKB activation in response to a raised glucose stimulus, which invokes GSIS and autocrine signalling by insulin in these cells. Although C2-ceramide appears not to modulate GSIS in our hands, recent work has suggested that the lipid does inhibit glucose-induced insulin gene expression in MIN6 b cells [38]. It is therefore possible that the effects of ceramide may be selectively targeted at insulin gene expression rather than hormone secretion, but given that we detect very little de novo synthesis of ceramide from palmitate the importance of such regulation may only become germane if the lipid is generated in significant amounts via other routes involving, for example, upregulation in the activity of membrane bound sphingomyelinases [39]. Whilst insulin is a very poor stimulus for inducing ERK activation in MIN6 b cells, cell incubation with high glucose (16 mM) leads to a robust activation/phosphorylation of ERK. Our findings indicate that activation of ERK signalling is important for supporting efficient GSIS/AASIS based on sensitivity to the MEK inhibitor, U0126. This inhibitor has been reported to promote activation of AMPK in HEK293 cells and so it is plausible the drug-induced reduction in GSIS/AASIS may be due to AMPK-mediated antagonism [40]. However, we believe this is highly unlikely given that U0126 has no direct effect on AMPK activity in cell-free assays [41] and does not promote any detectable increase in phosphorylation of acetyl CoA carboxylase, a physiological downstream AMPK substrate in MIN6 cells (data not shown). Previous work has shown that the sequalae of events that follow uptake and metabolism of glucose (i.e. increased cytoplasmic ATP/ADP ratio, closure of ATP-sensitive K+ channels, depolar- ization of the cell membrane, opening of voltage-gated calcium channels and the subsequent influx of extracellular calcium into the cell) induces activation of CaMKII, which acts upstream of MEK1/2 to mediate ERK phosphorylation [32,42]. ERK activation in turn has been reported to promote cytoskeletal rearrangement [43] and phosphorylation of cytoplasmic proteins implicated in exocytosis of insulin granules, such as synapsin I [44]. However, under conditions when b cells are chronically exposed to an elevated supply of palmitate the glucose-induced activation of ERK is significantly blunted. We believe that the reduced activation of ERK is likely to be a consequence of an associated reduction in CaMKII signalling. This belief is based not PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25975