EXPRESSION OF GLUT2 IN INSULIN-SECRETING ATT20 PITUITARY-CELLS

The importance of the glucose transporter isoform, GLUT2, in the const ruction of glucose-sensitive surrogate insulin-secreting cells was eva luated using murine pituitary AtT20 cells. The cells were double trans fected with cDNAs for human preproinsulin (hppI-1) driven by the cytom egalovirus promoter, and human GLUT2 driven by the beta-actin promoter . The stably transfected clone, AtTinsGLUT2.36, which strongly express ed both the hppI-1 and GLUT2 genes, constitutively released 7.5 ng/10( 6) cells/24 h of immunoreactive insulin-like material, 75% of which wa s fully processed mature human insulin. Increasing glucose concentrati ons in the subphysiological range up to 50 mu M increased insulin rele ase; but greater glucose concentrations did not further increase insul in release. Suppression of the low-K-m glucose-phosphorylating enzyme, hexokinase, with 2-deoxy-D-glucose increased glucose-stimulated insul in release by two- to threefold in the presence of subphysiological an d physiological glucose concentrations up to 10 mM. Physiological gluc ose concentrations increased the amount of GLUT2 mRNA, indicating that the beta-actin promoter responds in a glucose-dependent manner. Impla ntation of 2 x 10(7) AtTinsGLUT2.36 cells intraperitoneally into strep tozotocin-diabetic nude mice slowed the progression of hyperglycaemia. The implanted cells formed vascularised tumourlike cell aggregates at tached to the peritoneum. The results demonstrate that the beta-actin promoter is partially regulated by glucose. Expression of GLUT2 enable s glucose to enter the cell at high K-m, but high-K-m glucose phosphor ylation is also required to signal glucose-stimulated genes affecting insulin release.