The rat insulinoma cell line RIN 1046-38 loses glucose-stimulated insulin secretion as a function of time in culture. We found that the loss of glucose sensing in these cells was correlated with the loss of expression of GLUT-2 and glucokinase. Stable transfection of RIN cells with a plasmid containing the GLUT-2 cDNA conferred glucose-stimulated insulin release in intermediate but not high passage cells, with the near-maximal 3-fold increase occurring at 50 microM glucose. GLUT-2 expressing cells also exhibited a larger response to the combination of 5 mM glucose + 1 microM forskolin than untransfected cells (7.9 versus 1.6-2.7-fold, respectively). GLUT-2 expressing intermediate passage, but not high passage, RIN cells exhibited a 4-fold increase in glucokinase enzymatic activity relative to nonexpressing controls. Glucokinase activity was also increased by transfer of the GLUT-2 gene into intermediate passage RIN cells via recombinant adenovirus. Preincubation of GLUT-2 expressing intermediate passage RIN cells with 2-deoxyglucose to inhibit low Km hexokinases resulted in a glucose-stimulated insulin secretion response that was shifted toward the physiologic range. These studies indicate that GLUT-2 expression confers both a high and low affinity glucose-stimulated insulin secretion response to intermediate passage RIN cells.