In healthy pancreatic islets glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. nifedipine-sensitive calcium-channel flux. Following 3-to-11mM glucose stimulation all stressors substantially reduced the peak glucose-stimulated [Ca2+]i Hoechst 33342 response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor although FFAs showed a borderline reduction which contributed to a significant decrease in the stimulation index (11mM:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30mM KCl + 250uM diazoxide to test the amplifying pathway. Only Hoechst 33342 rotenone-treated islets showed a robust increase in 3-to-11mM glucose-stimulated insulin secretion under clamped conditions suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from COLL6 insulin secretion differently: ER stressors (thapsigargin cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs 28 rotenone) impacted insulin secretion. Keywords: islets beta-cells cytokines calcium insulin low-grade inflammation diabetes chronic free fatty acid palmitate oleate linoleate glucotoxicity high glucose endoplasmic reticulum thapsigargin unfolded protein response ER stress oxidative stress rotenone interleukin IL-1beta IL-6 GRAPHICAL ABSTRACT 1 Introduction The primary function of beta cells is to synthesize and secrete insulin a critical regulator of blood glucose. At the level of the individual beta-cell the ‘Consensus Model’ provides a detailed description of the cellular response to glucose stimulation as summarized in [1 2 In this model the beta cell is Hoechst 33342 electrically silent at low glucose concentrations (<~5 mM representing fasting conditions) secreting insulin at a low basal rate. In response to sharp increases in blood glucose beta cells take up glucose through glucose transporters. During this time the endoplasmic reticulum (ER) and mitochondria take up [Ca2+]i in response to increased glucose metabolism which causes an overall dip in [Ca2+]i. The resulting increase in the ATP to ADP ratio closes ATP-sensitive potassium channels (KATP-channels). As the dominant resting conductance of the beta cell KATP-channels normally hyperpolarize the beta-cell membrane under basal glucose conditions. The closure of KATP-channels in response to increased glucose metabolism depolarizes the cell membrane. This initiates the repetitive firing of calcium-dependent action potentials and the influx of calcium into the beta cell resulting in increased calcium in the mitochondria ER and nucleus. A large spike in calcium influx leads to the exocytosis of a readily releasable pool of docked insulin granules which is termed first phase insulin release. Following first phase insulin release [Ca2+]i and insulin secretion remain elevated throughout the second phase response for as long as glucose remains elevated. Because [Ca2+]i is a strong trigger of exocytosis both glucose-stimulated [Ca2+]i (GSCa) and glucose-stimulated insulin secretion (GSIS) show similar trajectories under these conditions. GSCa can thus be used to assess the physiological response of islets to glucose stimulation. [Ca2+]i imaging is advantageous because it provides high temporal precision of real-time changes in response to stimuli at the level of the individual islet . Changes in the latency trajectory and amplitude of the triphasic GSCa response may indicate Hoechst 33342 specific defects in stimulus-secretion coupling or other aspects of islet dysfunction. However there are also amplifying processes that operate in parallel with the pathways of the Consensus Model to couple glucose uptake and metabolism with insulin exocytosis [4-6]. The amplifying pathway allows additional insulin release to occur independently of changes in [Ca2+]i and thus provide a means by which the processes of insulin release and [Ca2+]i signaling can dissociate from one another. The exposure of islets to stress can further dissociate calcium signaling and insulin.