It has been previously demonstrated that the enteric hormone glucagon-like peptide-1 (7-36 amide) (GLP-1) has acute effects on glucose-induced insulin secretion by RIN 1046-38 cells. In this study, we investigated the effects of extended exposure of RIN 1046-38 cells to GLP-1 and examine the mechanism by which GLP-1 synergizes with glucose in stimulating insulin secretion. Compared with cells cultured with glucose alone, incubation of cells with glucose plus 1 or 10 nM GLP-1 for 12 or 24 h significantly increased insulin release by about 3-fold, intracellular insulin content by 1.5-fold, and insulin messenger RNA (mRNA) by almost 2.5-fold. The insulinotropic effects of GLP-1 on RIN 1046-38 cells were accompanied by an up-regulation of both glucose transporter-1 (GLUT-1) and hexokinase I mRNA by about 2-fold. mRNA levels of GLUT-2 and glucokinase, which were low in controls, were unchanged by GLP-1 treatment. Treatment of cells with a transcription inhibitor, actinomycin D, demonstrated that elevated insulin mRNA levels after a GLP-1 exposure are mainly due to stabilization of the mRNA. In contrast, the elevated mRNA levels of GLUT-1 and hexokinase I are the result of increased transcription stimulated by GLP-1 exposure. Actinomycin D blunted the GLP-1 effect on insulin release but did not affect GLP-1 mediated elevation of insulin mRNA. This suggests that actinomycin D inhibits the transcription of the proteins necessary for insulin biosynthesis and insulin release, such as GLUT-1 and hexokinase I. Our study suggests that the mechanisms by which extended exposure of RIN 1046-38 cells to GLP-1 increases glucose-stimulated insulin secretion include significant up-regulation of glucose-sensing elements.