Palmitate and Beta Cell Insulin Secretion Figure 3. The de novo ceramide synthesis pathway is not involved in the palmitate-induced loss in glucose-stimulated insulin secretion. MIN6 b cells were incubated either with or without 10 mM AZ422 (a SPT inhibitor) or 5 mM fenretinide (an inhibitor of dihydroceramide desaturase) (A) for 48 h or with varying concentrations of ceramide and dihydroceramide (B–F) for 2 h. Palmitate (0.4 mM) was present for 48 h where indicated. Glucose was added for 1 h for analysis of GSIS (A and B). Alternatively, cells were either lysed for immunoblot analysis using antibodies against (C) PKCf/lThr410 and total PKCf/l (used as loading control), (D) PKBSer473 and total PKB (as a loading control) or harvested and lipids extracted to assess (E) ceramide or (F) DAG content. Bars represent mean 6 SEM of 3 experiments and asterisks signify a significant difference from the untreated control value (P,0.05). doi:10.1371/journal.pone.0025975.g003 Carnitine provision has been shown to enhance fatty acid oxidation, whereas etomoxir targets carnitine palmitoyltransferase I (CPT1) and blocks mitochondrial fatty acid oxidation. Figure 7C shows that carnitine supplementation had no impact on palmitate’s inhibitory effect on GSIS, whereas blocking mitochon- drial fatty acid uptake using etomoxir caused a marginal enhancement in palmitate’s repressive effect on GSIS. Discussion Sustained increases in circulating free fatty acids is an early indicator of insulin resistance [33]. As with skeletal muscle and other insulin sensitive tissues, pancreatic beta cells are susceptible to the negative effects of fatty acid excess [34]. Whilst fatty acids are essential for supporting GSIS, increased availability as seen in PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25975