The establishment of surrogate islet beta cells is important for the treatment of diabetes. Hepatocytes have a similar glucose sensing system as beta cells and have the potential to serve as surrogate beta cells. In this report, we demonstrate that infection of Hepa1-6 liver cells with a lentivirus expressing the human insulin cDNA results in expression and secretion of human insulin. Furthermore, we show that L-arginine at low levels of glucose significantly stimulates the release of insulin from these cells, compared to exposure to high concentration of glucose. The arginine-induced insulin release is via the production of nitric oxide, since treatment with N-G-nitro-L-arginine, an inhibitor of nitric oxide synthase, blocks insulin secretion induced by L-arginine. These results indicate that nitric oxide plays a role in L-arginine-stimulated insulin release in hepatocytes expressing the human insulin gene, and provides a new strategy to induce insulin secretion from engineered non-beta cells. (c) 2005 Elsevier Inc. All rights reserved.