Aims/hypothesis: Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate aminotransferase (GFAT) as a rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine TGF-beta1, a key mediator in the development of diabetic nephropathy and possibly other diabetic angiopathies. In this study we investigated the effect of enhanced expression of wild-type GFAT and two enzymatically inactive GFAT mutants on TGF-beta1 synthesis in mesangial cells.
Methods: Mutated human GFAT expression vectors were prepared by PCR-site directed mutagenesis. Wild-type and mutated vectors were transfected into human embryonic kidney 293 cells and mesangial cells and GFAT enzyme activity was assessed by formation of glucosamine-6-phosphate. Production of TGF-beta1 and fibronectin protein was examined by ELISA.
Results: Mutation of histidine 577 or lysine 676 to alanine led to a complete loss of GFAT enzyme activity. An increased concentration of wild-type GFAT in mesangial cells enhanced both TGF-beta1 and fibronectin production 1.5-fold, while mesangial cells transfected with the mutated GFAT constructs showed no effect.
Conclusion/interpretation: The data indicate that the hexosamine pathway-mediated induction of TGF-beta1 synthesis in mesangial cells is dependent on GFAT enzyme activity. Our results add to previous observations showing that the hexosamine pathway could increase the transcriptional activity of nuclear proteins leading to enhanced cytokine synthesis.