Cellular morphology is one of the important factors for coordinating cell signaling. In this study, the morphological variation via glucose transporter (GLUT)-mediated anchoring was investigated in the cultures of human mammary epithelial cells in the presence or absence of insulin on culture surfaces with the changed ratios of d- and l-glucose displayed. With increasing ratio of d-glucose displayed on the surfaces, the cells showed a stretched shape in the culture with 10 mug/cm(3) insulin, reaching the highest extent of cell stretching at 100%d-glucose display, whereas round cells were predominant at 0%d-glucose display. In the absence of insulin, on the other hand, the extent of cell stretching showed a concave profile in terms of the ratio of d-glucose displayed, the extent being highest at 50%d-glucose display. Blocking of integrin alpha(5)beta(1) or GLUTs1 and 4 on the cells using corresponding antibodies revealed that the primary mechanism for cell attachment was based on integrin-mediated binding, and that GLUTs1 and 4 contributed largely to morphological changes of cells. Confocal microscopy further revealed that GLUT4 localization occurred in response to d-glucose display as well as insulin addition. In the absence of insulin, GLUT4 spots were extensively observed in the cell body regardless of whether d-glucose was displayed or not. However, in the presence of insulin, the broad distribution of GLUT4 appeared on the basal and apical sides of cells at 100%d-glucose display, in contrast with its localization only on the apical side of cells at 0%d-glucose display. These results suggest that the quantitative balance between GLUTs on the cytoplasmic membrane and d-glucose displayed on a culture surface determines the cell morphology, as explained by the receptor saturation model.