Aim To investigate the acute and chronic effects of l-leucine on pancreatic a-cell function in vitro. Furthermore, we wanted to explore if glucagon-like peptide-1 (GLP-1), isosteviol (ISV) and 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR) counteract changes in alpha-cell function induced by chronic exposure to leucine. Methods Isolated mice islets were incubated with 10 mM leucine for 2 or 72 h. We investigated glucagon and insulin secretion at 2 mM and 16.7 mM glucose. In addition, we cultured clonal alpha-TC1-6 cells with 5 mM leucine, 5 mM leucine plus GLP-1 (10-6 M), or ISV (10-6 M) or AICAR (10-5 M) at high glucose for 72 h. We measured the glucagon secretion, cholesterol (CHO) and triglyceride (TG) content, cell proliferation as well as gene expression. Results Ten millimolar of leucine for 2 h significantly stimulated glucagon and insulin secretion both at 2 and 16.7 mM glucose in mice islets. After 72 h incubation with 10 mM leucine the glucagon secretion was enhanced at both 2 and 16.7 mM glucose, whereas the glucose-stimulated insulin secretion (16.7 mM glucose) was inhibited. Chronic exposure to 5 mM leucine increased glucagon secretion, CHO and TG content, cell proliferation and Pcsk2 (p < 0.001), MafB (p < 0.05), Gcg (p < 0.001), Prkaa1 (p < 0.01), Hmgcr (p < 0.001), Srebf2 (p < 0.001), Acaca (p < 0.001), Mtor (p < 0.05) mRNA expression in clonal alpha-TC1-6 cells. While GLP-1 was cable of reducing glucagon hypersecretion and Pcsk2 (p < 0.05) mRNA expression. ISV and AICAR had no effect on leucine-induced glucagon hypersecretion. Conclusions Long-term exposure to leucine induces hypersecretion of glucagon secretion, that is, aminoacidotoxicity and influences some key genes of pancreatic alpha-cells. Interestingly, GLP-1 counteracts the leucine-induced a-cell dysfunction.