The hormone glucose-dependent insulinotropic polypeptide (GIP) is an important regulator of insulin secretion. GIP has been shown to increase adenylyl cyclase activity, elevate intracellular Ca(2+) levels, and stimulate a mitogen-activated protein kinase pathway in the pancreatic beta-cell. In the current study we demonstrate a role for arachidonic acid in GIP-mediated signal transduction. Static incubations revealed that both GIP (100 nm) and ATP (5 microm) significantly increased [(3)H]arachidonic acid ([(3)H]AA) efflux from transfected Chinese hamster ovary K1 cells expressing the GIP receptor (basal, 128 +/- 11 cpm/well; GIP, 212 +/- 32 cpm/well; ATP, 263 +/- 35 cpm/well; n = 4; p < 0.05). In addition, GIP receptors were shown for the first time to be capable of functionally coupling to AA production through Gbetagamma dimers in Chinese hamster ovary K1 cells. In a beta-cell model (betaTC-3), GIP was found to elicit [(3)H]AA release, independent of glucose, in a concentration-dependent manner (EC(50) value of 1.4 +/- 0.62 nm; n = 3). Although GIP did not potentiate insulin release under extracellular Ca(2+)-free conditions, it was still capable of elevating intracellular cAMP and stimulating [(3)H]AA release. Our data suggest that cAMP is the proximal signaling intermediate responsible for GIP-stimulated AA release. Finally, stimulation of GIP-mediated AA production was shown to be mediated via a Ca(2+)-independent phospholipase A(2). Arachidonic acid is therefore a new component of GIP-mediated signal transduction in the beta-cell.