Chronic Effects of Palmitate Overload on Nutrient- Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells Maria L. Watson 1 , Katherine Macrae1, Anna E. Marley2, Harinder S. Hundal1* 1 Division of Cell Signalling and Immunology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, United Kingdom, 2 AstraZeneca, CVGI, Alderley Park, Macclesfield, Cheshire, United Kingdom Abstract Background: Sustained exposure of pancreatic b cells to an increase in saturated fatty acids induces pleiotropic effects on b- cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling. Principal Findings: GSIS and AASIS were both stimulated by .7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin- dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (,75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate. Conclusions: Our results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 b cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion. Citation: Watson ML, Macrae K, Marley AE, Hundal HS (2011) Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells. PLoS ONE 6(10): e25975. doi:10.1371/journal.pone.0025975 Editor: Irina Agoulnik, Florida International University, United States of America Received June 17, 2011; Accepted September 14, 2011; Published October 5, 2011 Copyright: 2011 Watson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MW and KM have been supported by Biotechnology and Biological Sciences Research Council-AstraZeneca CASE scholarships. The authors are grateful for financial support from the European Commission (contract LSHM-CT-20004-005272) and Diabetes United Kingdom. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: One of the authors, AM, is an employee of AstraZeneca and two of the other authors, MW and KM, have been supported by funds from AstraZeneca. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: h.s.hundal@dundee.ac.uk Introduction Hyperlipidemia is one of a cluster of abnormalities associated with the metabolic syndrome, which not only promotes insulin resistance, but results in the dysfunction of numerous cellular responses in tissues such as skeletal muscle [1], heart [2], liver [3], adipose [4] and the pancreas [5]. In the pancreas, for example, it has been demonstrated that during the pre-diabetic and diabetic states, there is an increase in intracellular fatty acids [6] that desensitise pancreatic beta cells to glucose [7]. An important consequence of this reduced glucose ‘‘sensing’’ capacity is a reduction in glucose-stimulated insulin secretion (GSIS), which contributes to the impaired glucose homeostasis associated with the diabetic state. Fatty acids are thought to play an essential role in GSIS augmenting the glucose-induced secretion of insulin [8,9]. During the fasting state, fatty acids are free in the cytosol of beta cells, and, under these circumstances, are channelled into mitochondria for b-oxidation and generation of ATP [10] and do not promote any detectable increase in insulin secretion. However, upon feeding, the rise in blood glucose not only promotes insulin release from beta cells by a mechanism involving the inactivation (closure) of plasma membrane K+ATP channels, but glucose will also contribute to metabolic anaplerosis. Citrate produced in the mitochondria from glucose metabolism will form malonyl-CoA in the cytosol [11,12], which prevents b-oxidation by inhibiting carnitine palmitoyltransferase (CPT-1), thereby allowing an increase in long chain fatty acids which can stimulate insulin secretion. Although the precise mechanism is unknown it is thought that long chain fatty acids can either modulate intracellular targets that stimulate insulin release [13,14] or form complex lipids, such as diacylglycerol (DAG), that interact with insulin granule proteins leading to granule fusion with the membrane [15]. In addition, the presence of free fatty acids, PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25975