Transcriptional regulation of transforming growth factor β1 by glucose:: Investigation into the role of the hexosamine biosynthesis pathway

Background: The hexosamine biosynthesis pathway (HBP) is hypothesized to mediate many of the adverse effects of hyperglycemia. We have shown previously that increased flux through this pathway leads to induction of the growth factor transforming growth factor-alpha (TGF-alpha) and to insulin resistance in cultured cells and transgenic mice. TGF-beta is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore hypothesized that the HBP was involved in the regulation of TGF-beta by glucose in rat vascular and kidney cells. Methods: A plasmid containing the promoter region of TGF-beta 1 cloned upstream of the firefly luciferase gene was electroporated into rat aortic smooth muscle, mesangial, and proximal tubule cells. Luciferase activity was measured in cellular extracts from cells cultured in varying concentrations of glucose and glucosamine. Results: Glucose treatment of all cultured cells led to a time- and dose-dependent stimulation 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 until 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 increase in TGF beta 1-transcriptional activity. The stimulatory effect of glucosamine was also dose-dependent but was slower to develop and longer lasting than 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.