OBJECTIVE: The close relationship between obesity and type 2 diabetes (T2D) highlights the fact that most diabetes patients are overweight or obese. We propose that elevated glucose and free fatty acid levels in those patients cause beta-cell dysfunction. Chronic exposure to excess nutrients (glucose and free fatty acid) leads to glucolipotoxicity, characterized by basal insulin hypersecretion, a left-shift in the glucose dose-dependent insulin secretion curve, and blunted glucose-stimulated insulin secretion. One of the suggested reasons for this defect is elevated intracellular lipid. In this study, our objective was to investigate whether reducing beta-cell lipid levels can reverse basal insulin hypersecretion. METHODS: INS-1 (823/13) cells were cultured in 4 or 11 mM glucose media. Elevated glucose and KCl doses were added to cells in the insulin secretion experiments. In the KCl-induced insulin secretion experiment, cells were treated with a combination of 12 mM glucose and 250 μM diazoxide, then assigned to different test concentrations with elevated KCl doses. Insulin release and content were measured by the insulin ultra-sensitive homogenous time-resolved fluorescence (HTRF) kit (Cisbio). Following that, we monitored intracellular Ca2+ activity of KCl-induced insulin secretion on a fluorescence spectrophotometer F-2000 (Hitachi). Additionally, we acutely added Adipo C (20 µM) or fatty acid-free BSA to cells to reduce the lipids levels in the ß-cells. We also stained with Nile Red (Sigma) to examine the intrinsic lipid droplets in those cells. RESULTS: ß-cells cultured in excess nutrients (11 mM glucose) exhibited a left shift in the glucose dose-dependent response curve. The hypersecretion at low glucose could be blocked by the KATP channel activator, diazoxide, indicating that Ca2+ influx drives the increase in secretion at glucose concentrations normally considered basal. Here we extend this left shift to include KCl-induced insulin secretion, supporting a role for Ca2+ in the observed hypersensitivity. KCl-induced Ca2+ influx was also left-shifted. Interestingly, we found acute exposure to Adipo C or fatty acid-free BSA reversed the basal hypersecretion in cells cultured in excess nutrients. CONCLUSION: The work presented in this study provided supporting evidence that ß-cells cultured in excess nutrients were hypersensitive to glucose while extending these studies to KCl-induced insulin release. The excess nutrient-induced left shift in both glucose and KCl-stimulated insulin secretion was mediated by increased Ca2+. Thus, we postulate that excess nutrient exposure increases ß-cell plasma membrane lipids that alter Ca2+ handling to allow increased Ca2+ influx at inappropriate low glucose concentrations. Our results demonstrated that cells acutely exposed to the putative long-chain acyl-CoA synthetase inhibitor Adipo C or fatty acid-free BSA reversed basal insulin hypersecretion and supports a role for lipids mediating the adverse effect. T2D patients with obesity have a similar physiologically elevated fasting blood glucose and lipid. Thus, our findings suggest lowering lipid levels in ß-cells may have therapeutic potential in treating hyperinsulinemia leading to T2D.