Regulation of glutamine:fructose-6-phosphate amidotransferase activity by high glucose and transforming growth factor beta in rat mesangial cells

Background: The hexosamine biosynthesis pathway acts as a cellular glucose sensor and mediates many of the adverse effects of glucose. Increased flux through this pathway results in insulin resistance in rat fibroblasts and transgenic mice and upregulation of transforming growth factor beta (TGF-beta) transcriptional activity in rat kidney cells, The first and rate-limiting step in this pathway, which is responsible for the metabolism of glucose to glucosamine, is catalyzed by glutamine:fructose-6-phosphate amidotransferase (GFA), Methods: Because of the known effects of hyperglycemia on mesangial cell (MC) function and growth factor regulation, we examined the regulation of GFA by glucose and TGF-beta in cultured SV40 rat MCs, GFA activity was assayed in cytosolic extracts of MCs using high-performance liquid chromatography, Results: Culturing in 10 and 25 mM of glucose for 24 hours resulted in 33.4% (P<0.025) and 43.5% (P<0.05) decreases in GFA activity when compared with cells cultured at 1 to 5 mM of glucose. The downregulation in GFA activity by high glucose (HG) required at least 6 hours in culture and persisted for several days, HG effects were not a result of osmolar changes or glucose-induced differences in glucose uptake, Like HG, treatment of MCs with TGF-beta (2 ng/mL) for 4 hours resulted in a 30% (P<0.05) decrease in GFA activity in cells cultured at 1 mM glucose, but the effects of TGF-<beta> were not additive to those of HG, TGF-beta -mediated downregulation of GFA activity was inhibited by a TGP-beta -neutralizing antibody, but HG's effects were not. Insulin-like growth factor-1 (IGF-I) had similar effects as TGF-beta, but GFA activity was not regulated by angiotensin II. Conclusions: GFA activity is downregulated by HG, TGF-beta, and IGF-1 in rat MCs, Downregulation of this cellular glucose sensor may be a protective mechanism against the harmful effects of excess glucose as seen in diabetes.