Background: Elevated glucose may cause β-cell dysfunction in type 2 diabetes, i.e., glucotoxicity. Results: Elevated glucose, but not pyruvate, perturbed insulin secretion and content, plasma and mitochondrial membrane potentials, and proton leak, while increasing glycolytic metabolites in β-cells. Conclusion: Early metabolism of glucose exerts a toxic effect on clonal insulin-producing cells. Significance: Unraveling these mechanisms may provide protection of β-cells in diabetes. Glucotoxicity in pancreaticβ-cells is a well established pathogenetic process in type 2 diabetes. It has been suggested that metabolism-derived reactive oxygen species perturb the β-cell transcriptional machinery. Less is known about altered mitochondrial function in this condition. We used INS-1 832/13 cells cultured for 48 h in 2.8 mM glucose (low-G), 16.7 mM glucose (high-G), or 2.8 mM glucose plus 13.9 mM pyruvate (high-P) to identify metabolic perturbations. High-G cells showed decreased responsiveness, relative to low-G cells, with respect to mitochondrial membrane hyperpolarization, plasma membrane depolarization, and insulin secretion, when stimulated acutely with 16.7 mM glucose or 10 mM pyruvate. In contrast, high-P cells were functionally unimpaired, eliminating chronic provision of saturating mitochondrial substrate as a cause of glucotoxicity. Although cellular insulin content was depleted in high-G cells, relative to low-G and high-P cells, cellular functions were largely recovered following a further 24-h culture in low-G medium. After 2 h at 2.8 mM glucose, high-G cells did not retain increased levels of glycolytic or TCA cycle intermediates but nevertheless displayed increased glycolysis, increased respiration, and an increased mitochondrial proton leak relative to low-G and high-P cells. This notwithstanding, titration of low-G cells with low protonophore concentrations, monitoring respiration and insulin secretion in parallel, showed that the perturbed insulin secretion of high-G cells could not be accounted for by increased proton leak. The present study supports the idea that glucose-induced disturbances of stimulussecretion coupling by extramitochondrial metabolism upstream of pyruvate, rather than exhaustion from metabolic overload, underlie glucotoxicity in insulin-producing cells.