INSULIN-SECRETION, INSULIN CONTENT AND GLUCOSE PHOSPHORYLATION IN RINM5F INSULINOMA CELLS AFTER TRANSFECTION WITH HUMAN GLUT2 GLUCOSE-TRANSPORTER CDNA

The insulin-secretory response to glucose is defective in the RINm5F i nsulin-producing tumour cell line. Stable transfection with human low- affinity GLUT2 glucose-transporter cDNA revealed a significant improve ment in stimulus-secretion coupling in these insulinoma cells. 3-O-Met hylglucose uptake increased 10-fold in the concentration range. 10-20 mM, Whereas non-transfected control cells were unresponsive. Northern- blot analysis revealed a 7-fold increase in expression of the insulin gene in the GLUT2-transfected RINm5F cell clone T1. In contrast, gluco kinase and GLUT1 glucose-transporter mRNA gene expression were not aff ected by transfection with GLUT2 glucose-transporter cDNA. The insulin content of transfected RINm5F cells was 7-fold higher after tissue cu lture at high glucose concentrations than in non-transfected controls. GLUT2-transfected RINm5F cells also regained insulin-secretory respon siveness toward high glucose concentrations. Tissue culture for 72 h i n 20 mM glucose induced glucokinase activity in the GLUT2-transfected RINm5F clone TI, raising the glucokinase/hexokinase phosphorylation ra tio from 0,2 to 0.6. The experiments demonstrate that an increased glu cose uptake via a low-affinity glucose transporter and an increased me tabolic flux rate are important factors in the induction of insulin-ge ne expression and glucokinase activity and thus improved glucose-induc ed biosynthesis and secretion of insulin in RINm5F insulinoma cells.