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 t ransgenic mice and upregulation of transforming growth factor beta (TGF-bet a) transcriptional activity in rat kidney cells, The first and rate-limitin g step in this pathway, which is responsible for the metabolism of glucose to glucosamine, is catalyzed by glutamine:fructose-6-phosphate amidotransfe rase (GFA), Methods: Because of the known effects of hyperglycemia on mesangial cell (M C) 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 fo r 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-b eta (2 ng/mL) for 4 hours resulted in a 30% (P<0.05) decrease in GFA activi ty in cells cultured at 1 mM glucose, but the effects of TGF-<beta> were no t additive to those of HG, TGF-beta -mediated downregulation of GFA activit y 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 ra t MCs, Downregulation of this cellular glucose sensor may be a protective m echanism against the harmful effects of excess glucose as seen in diabetes.