Background: Elevated glucose may cause-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. [ABSTRACT FROM AUTHOR]