NONENZYMATIC GLYCOSYLATION IN-VITRO AND IN BOVINE ENDOTHELIAL-CELLS ALTERS BASIC FIBROBLAST GROWTH-FACTOR ACTIVITY - A MODEL FOR INTRACELLULAR GLYCOSYLATION IN DIABETES

Intracellular sugars are more reactive glycosylating agents than gluco se. In vitro nonenzymatic glycosylation of basic fibroblast growth fac tor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3 -phosphate (G3P) reduced high affinity heparin-binding activity of rec ombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosyla tion in GM7373 endothelial cells, we first demonstrated that GLUT-1 tr ansporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, a nd the intracellular glucose concentration increased sixfold at 24 h a nd fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation endproducts (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 mM glucose, demonstrating that al l the mitogenic activity was due to bFGF. Immunoblotting and ELISA sho wed that 30 mM glucose did not decrease detectable bFGF protein, sugge sting that the marked decrease in bFGF mitogenic activity resulted fro m posttranslational modification of bFGF induced by elevated glucose c oncentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. Thes e data are consistent with the hypothesis that nonenzymatic glycosylat ion of intracellular protein alters vascular cell function.