Presence of glutamine : fructose-6-phosphate amidotransferase for glucosamine-6-phosphate synthesis in endothelial cells: effects of hyperglycaemia and glutamine

Aims/hypothesis. Recent studies show that glucosamine infusion impairs endo thelium-dependent blood flow in normoglycaemic rats. The pathophysiological relevance of this finding, however, depends on whether de novo glucosamine synthesis occurs in endothelial cells. The aim of this study was to test t he hypothesis of whether glutamine:fructose-6-phosphate amidotransferase (t he first and key regulatory enzyme in hexosamine synthesis) is present for endothelial glucosamine synthesis. Methods. Bovine venular, bovine aortic, human microvascular, human umbilica l vein, and rat coronary microvascular endothelial cells were used to measu re glutamine:fructose-6-phosphate amidotransferase activity. To determine, glucosamine-6-phosphate synthesis in intact cells, they were incubated for 1 h in Krebs bicarbonate buffer containing 5, 15 or 30 mmol/l (U-C-14] gluc ose and 0.5, 2 or 4 mmol/l glutamine. The [C-14] Glucosamine-6-phosphate an d its end products ([C-14]UDP-N-acetylglucosamine and [C-14]UDP-N-acetylgal actosamine) were separated by HPLC. Results. There were high glutamine:fructose-6-phosphate amidotransferase ac tivities in all endothelial cells studied. Exposure of cells to 15 to 30 mm ol/l glucose or 2 to 4 mmol/l glutamine increased enzyme activity. Glucosam ine-6-phosphate, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine synt heses increased with increasing extracellular concentrations of glucose fro m 5 to 30 mmol/l or of glutamine from 0.5 to 4 mmol/l. Conclusion/interpretation. Our results show the presence of glutamine:fruct ose-6-phosphate amidotransferase for de novo glucosamine synthesis in endot helial cells and the modulation of this pathway by hyperglycaemia and gluta mine. As glucosamine inhibits endothelial nitric oxide synthesis, these fin dings could have important implications for impaired endothelium-dependent relaxation and vascular dysfunction in diabetes mellitus.