Glucose metabolism to glucosamine is necessary for glucose stimulation of transforming growth factor-alpha gene transcription

Abstract

Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of glucose, and this effect of glucose can be mimicked by glucosamine. To determine whether the metabolism of glucose to glucosamine is required for this glucose effect, the rate-limiting step in glucose metabolism to glucosamine through the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) was blocked using pharmacological and antisense strategies. We found that blockage of GFAT activity or expression significantly blunted the glucose-induced increase of TGFalpha expression. Blockage of GFAT also resulted in a decreased RL2 signal on intracellular proteins as detected by Western blotting and indirect immunofluorescence. The RL2 monoclonal antibody recognizes an epitope on proteins that contain N-acetylglucosamine and thus is a measure of protein glycosylation. Conversely, treatment of the cells with glucose and glucosamine resulted in an increase in the RL2 epitope on intracellular proteins. These results indicate that the metabolism of glucose to glucosamine is necessary for the transcriptional stimulation of TGFalpha expression in vascular smooth muscle cells by glucose. Furthermore, the level of glycosylation of some intracellular proteins can be modulated in response to physiological changes in the extracellular glucose concentration and the net activity of GFAT.