Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic p-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in similar to 30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin I A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion. (C) 2009 Elsevier Inc. All rights reserved.