Palmitate and Beta Cell Insulin Secretion Figure 5. ERK inhibition induces a loss in glucose-stimulated insulin secretion (GSIS) and amino acid-stimulated insulin secretion (AASIS). MIN6 b cells were incubated in the absence or presence of 20 mM U0126, as indicated prior to being exposed to either 3 mM or 16 mM glucose for 1 h or alternatively to an amino acid stimulus (10 mM leucine/glutamine and 3 mM glucose). At the end of this incubation cells were (A) lysed for analysis of ERK phosphorylation in whole cell lysates or (B) prepared for analysis of net changes in GSIS or AASIS (B) as described in methods. 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.g005 only on the finding that palmitate impairs the glucose-induced activation of CaMKII, but the observation that, even in cells not exposed to the fatty acid, CaMKII inhibition with KN-62 suppresses activation of ERK in response to a raised glucose stimulus. Moreover, consistent with the idea that CaMKII- directed signaling is important for promoting insulin secretion we find that KN-62 causes a significant reduction in GSIS. In addition to glucose, certain amino acids are also potent insulin secretagogues being able to induce hormone secretion as a consequence of their capacity to depolarise the b-cell membrane and cause the concomitant opening of voltage-gated calcium channels. Membrane depolarisation may be triggered directly by some amino acids (e.g. arginine), whereas others (e.g. alanine, glutamine, leucine) may induce it via a combination of their influx into b-cells via Na-coupled amino acid transporters and/or metabolism that enhances ATP generation with subsequent effects upon K-ATP channels in much the same manner as glucose [45]. Given that the effects of glucose and amino acids converge upon regulation of a common plasma membrane channel it is perhaps not unsurprising that sustained exposure of MIN6 b cells to palmitate also results in impaired downstream activation of CaMKII and ERK in response to amino acid provision. A key issue of interest is the mechanism by which over supply of palmitate induces impaired GSIS. Since metabolism of glucose and the associated increase in cytoplasmic ATP/ADP ratio is central for stimulus-secretion coupling in b-cells it is plausible that palmitate may reduce glucose metabolism and thereby limit GSIS. However, previous work has shown that sustained (72 h) incubation of MIN6 b cells with palmitate does not affect glycolytic flux or ATP synthesis [46] negating this as a likely possibility. It also appears unlikely that oxidation of palmitate itself is required for initiating the reduction in GSIS. Etomoxir, which inhibits mitochondrial fatty acid uptake via CPT1, failed to prevent the reduction in GSIS, whereas carnitine supplementation, which would promote mitochondrial fatty acid uptake/oxidation [47], did not augment the palmitate-induced loss in insulin secretion. Furthermore, the finding that 2-bromopalmitate, which cannot undergo mitochondrial oxidation, was as potent as palmitate in suppressing GSIS underscores the notion that changes in mitochondrial fatty acid oxidation are unlikely to account for the observed reduction in GSIS. This latter observation is also in line with work assessing the inhibitory effect of palmitate on glucose-induced insulin gene expression in b cells in which 2- bromopalmitate was found to mimic the repressive effect of palmitate and in which fatty acid esterification rather than oxidation was deemed as the more important determinant of palmitate-induced lipotoxicity [48]. Consistent with this suggestion we did observe that the repressive effect of palmitate upon GSIS was further enhanced in the presence of etomoxir, which may promote greater partitioning of palmitate into pathways favouring its esterification to diacylglycerol (DAG) and triglyceride. While we did not detect significant generation of ceramide from palmitate in PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25975