Palmitate and Beta Cell Insulin Secretion Figure 6. Palmitate antagonises the stimulus-induced activation of ERK and CaMKII in MIN6 b cells. MIN6 b cells were incubated with 10 mM KN62 (an inhibitor of CaMKII) for 2 h. In the last hour of this incubation cells were incubated with either glucose (16 mM) or amino acids (10 mM) prior to assessing (A) GSIS or AASIS, (B) ERK activation by analysis of ERKThr202/Tyr204 phosphorylation. Total ERK content was used for assaying protein loading. Alternatively, MIN6 b cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h and then stimulated insulin secretion assessed by the addition of glucose or amino acids for 1 h at the concentrations indicated. Cells were lysed and lysates immunoblotted to assay phospho-CaMKIIThr286 phosphorylation with native CaMKII serving as a gel loading control (C). 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.g006 MIN6 b cells synthesis of DAG was indeed elevated in palmitate- treated MIN6 b cells. An increase in intracellular DAG would be expected to promote activation of DAG-sensitive PKCs, which are thought to mediate the effects of acute increases in fatty acid provision upon numerous aspects of b-cell function, including potentiation of GSIS [49]. However, it is plausible that sustained fatty acid overload may result in synthesis and accumulation of distinct DAG species and/or activation of specific PKC isoforms that are potentially pathogenic in nature. Cantley and coworkers have suggested that PKCe may contribute to impaired GSIS given that its deletion in mouse b-cells induces amplification of pathways stimulating GSIS in isolated islets cultured chronically in the presence of palmitate [50]. Whilst the increase in GSIS induced in PKCe knock-out islets was suggested to operate independently of any changes in intracellular calcium, very recent work has reported that sustained exposure of islets to palmitate impairs calcium influx at membrane domains that are in close proximity to insulin granules with a consequential reduction in their exocytosis [51]. Using a fluorescent calcium indicator dye, Oregon-Green- BAPTA-1, we find that glucose induces calcium transients in MIN6 b cells and that these are muted in cells that have been chronically pre-exposed to palmitate for 48 hours (see Figure S2). It is plausible that these calcium transients may normally support the activation of CaMKII and that their reduction impairs efficient PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25975