Transcriptional regulation of transforming growth factor beta 1 by glucose: Investigation into the role of the hexosamine biosynthesis pathway

Background: The hexosamine biosynthesis pathway (HBP) is hypothesized to me diate many of the adverse effects of hyperglycemia. We have shown previousl y that increased flux through this pathway leads to induction of the growth factor transforming growth factor-alpha (TGF-alpha) and to insulin resista nce in cultured cells and transgenic mice. TGF-beta is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore h ypothesized that the HBP was involved in the regulation of TGF-beta by gluc ose in rat vascular and kidney cells. Methods: A plasmid containing the pro moter region of TGF-beta 1 cloned upstream of the firefly luciferase gene w as electroporated into rat aortic smooth muscle, mesangial, and proximal tu bule cells. Luciferase activity was measured in cellular extracts from cell s cultured in varying concentrations of glucose and glucosamine. Results: G lucose treatment of all cultured cells led to a time- and dose-dependent st imulation in TGF-beta 1 transcriptional activity, with high (20 mM) glucose causing a 1.4- to 2.0-fold increase. Glucose stimulation did not occur unt il after 12 hours and disappeared after 72 hours of treatment. Glucosamine was more potent: than glucose, with 3 mM stimulating up to a 4-fold increas e in TGF beta 1-transcriptional activity. The stimulatory effect of glucosa mine was also dose-dependent but was slower to develop and longer lasting t han that of glucose. Conclusions: The metabolism of glucose through the HBP mediates extracellular matrix production, possibly via the stimulation of TGF-beta in kidney cells. Hexosamine metabolism therefore, may play a role in the development of diabetic nephropathy.