Aims/Hypothesis: The aims of this study were to elucidate long-term effects of increased fatty acids and glucose concentrations on islet hormone secretion, triglyceride (TG) accumulation and fuel metabolism, and to determine the role of insulin on glucagon secretion. Methods: Isolated normal mouse islets were exposed to palmitate (0.6 mM) in the presence of high glucose (16.7 mM). After 48 h culture, glucagon secretion and content, insulin secretion and content, TG content and glucose oxidation were measured. The impact of etomoxir, an inhibitor of carnitine palmitoyl transferase-1, as well as of insulin, and alterations in gene expression were also investigated. Results: In the presence of palmitate, (i) high glucose caused no statistically significant suppression of glucagon while this was seen in the absence of palmitate; (ii) the insulin response to high glucose was impaired and (iii) an accumulation of TG and a decline in glucose oxidation were detected, whereas the glucagon content remained unchanged. However, etomoxir was capable of reducing glucagon secretion. Addition of exogenous insulin (10(-1) -10(-6) M) failed to restore alpha cell response to normal. Furthermore, 0.6 mM palmitate reduced the mRNA levels of acetyl-CoA carboxylase-1 and sterol regulatory element-binding protein-1c. Conclusions/Interpretation: In summary, high concentrations of palmitate and glucose cause a relative increase in glucagon secretion, a decline in insulin secretion, a loss of alpha cell sensitivity to glucose and an accumulation of TG. The inability of insulin to suppress glucagon may be because of insulin resistance of alpha cells.