Leukocyte type 12-lipoxygenase (12-LO) is an enzyme specifically expressed in the beta cells of the pancreas. 12-LO oxidizes fatty acids such as arachidonic acid and linoleic acids to their respective hydroperoxides. Increased concentration of lipid hydroperoxides causes oxidative stress and this could lead to cellular dysfunction. Increased expression of 12-LO in beta cells has been observed with use of inflammatory cytokines and during the prediabetic phase of beta cell dysfunction in the Zucker diabetic fatty rat model. Also mice deficient in 12-LO expression show a decreased incidence of immune-mediated diabetes. To further understand the role of 12-LO in beta cell metabolism, we over-expressed mouse leukocyte type 12-LO in INS-1 cells (transformed rat beta cell line) using an adeno-associated virus (AAV) vector system. In 12-LO over-expressing cells, cell-associated 12-HETE levels increased approximately 5- and approximately 3-fold in the culture supernatant. In the cells over-expressing 12-LO, glucose-stimulated insulin secretion (GSIS) decreased by 25-30% one hour after exposure to high glucose (15mM). By 2h, GSIS decreased by 50-54% at high glucose levels. These data suggest that increased 12-LO products can reduce the synthesis, processing or secretion of insulin in beta cells. We next examined the effect of 12-LO over-expression on mitogen-activated protein kinases (MAPK) by Western blot analyses using antibodies specific for different phospho-MAP kinases. Over-expression of 12-LO led to an activation of c-Jun N-terminal kinase while it markedly reduced Erk1 and 2 phosphorylation (4-fold). Further, over-expression of 12-LO led to induction of apoptosis in beta cells as determined by DNA ladder assay. These results suggest that increased 12-LO plays a key role in altering beta cell metabolism. Thus, increased 12-LO expression can have a detrimental effect on pancreatic beta cell function and viability, suggesting that blockade of 12-LO activity or expression could provide a novel way to protect beta cells from inflammatory injury.