The G-protein-coupled receptor GPR40 is specifically expressed in pancreatic beta-cells and has been identified as a long-chain fatty acid receptor (1;2). Since fatty acids do not initiate insulin secretion in the absence of glucose but strongly potentiate glucose-stimulated insulin secretion, GPR40 has received considerable interest as a potential therapeutic target for type 2 diabetes. Several pharmaceutical companies have active programs towards that goal, including one recently completed phase II trial (3). Previous work performed in our laboratory has demonstrated that GPR40 mediates approximately 50% of the effect of fatty acids in potentiating glucose-induced insulin secretion (4). Deletion of GPR40 renders mice more susceptible to high-fat-diet induced diabetes (5), indicating that the receptor is important for beta-cell compensation for insulin resistance and supporting the concept that GPR40 agonists have therapeutic potential in type 2 diabetes. In a recent study we observed that glucose stimulates GPR40 gene transcription in pancreatic beta-cells via increased binding of pancreas-duodenum homeobox-1 (Pdx-1) to the A-box in the HR2 region of the GPR40 promoter (6). Mutation of the Pdx-1 binding site within the HR2 nearly abolishes glucose activation of GPR40 promoter activity. The stimulation of GPR40 expression and Pdx-1 binding to the HR2 in response to glucose are mimicked by N-acetyl glucosamine, an intermediate of the hexosamine biosynthesis pathway, and involve phosphatidylinositol3-kinase-dependent O-GlcNAcylation of Pdx-1 in the nucleus. We demonstrated that O-GlcNAc transferase (OGT), the enzyme that modifies proteins by O-GlcNAcylation, interacts with the product of the PI3K reaction, phosphatidylinositol 3,4,5-trisphosphate (PIP3), in the nucleus. This interaction enables OGT to catalyze O-GlcNAcylation of nuclear proteins, including Pdx-1. We concluded from this study that glucose stimulates GPR40 gene expression at the transcriptional level through Pdx-1 binding to the HR2 region and via a signaling cascade that involves an interaction between OGT and PIP3 at the nuclear membrane (6). Activation of GPR40 by fatty acids does not appear to modulate intracellular fuel metabolism in islets (7), but triggers phospholipase C-mediated hydrolysis of membrane phospholipids downstream of Gαq. We have recently completed a study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids (Ferdaoussi M et al., manuscript in preparation). We observed that the fatty acid oleate potentiates the second-phase of glucose-stimulated insulin secretion in perifused islets, and that this effect is largely dependent upon GPR40. Accordingly, oleate induces F-actin remodeling in wild-type but not in GPR40-/- islets. Oleate induces phosphorylation of protein kinase D at residues Ser-744/748 and Ser-916 in wild-type but not GPR40-/- islets. Importantly, oleate potentiation of glucose-induced insulin secretion is lost upon pharmacological inhibition or deletion of pkd1. We concluded from this study that the signaling cascade downstream of GPR40 activation by fatty acids involves activation of protein kinase D, F-actin depolymerization, and potentiation of second-phase insulin secretion. Collectively, these findings provide important mechanistic information on the biology of GPR40, a novel drug target for type 2 diabetes.