Flux Through the Hexosamine Pathway Is a Determinant of Nuclear Factor κB– Dependent Promoter Activation

Abstract

The hexosamine pathway may mediate some of the toxic effects of glucose. We hypothesized that flux through this pathway might regulate the activity of nuclear factor κB (NF-κB)-dependent genes in mesangial cells (MCs). In MCs, RT-PCR revealed that high glucose (30 mmol/l) and glucosamine (1 mmol/l) increased mRNA levels for vascular cell adhesion molecule 1 (VCAM-1) and increased the activity of an NF-κB enhancer by 1.5- and 2-fold, respectively. Overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for flux through the hexosamine pathway, led to a 2.2-fold increase in NF-κB enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 μmol/l O-diazoacetyl-l-serine (azaserine) or 6-diazo-5-oxonorleucine. High glucose, glucosamine, and GFAT overexpression increased binding of MC nuclear proteins to NF-κB consensus sequences. Immunoblotting revealed that the p65 subunit of NF-κB was O-glycosylated in MC cultured in physiologic glucose and that significant enhancement occurred with high glucose and glucosamine. Both glucose and glucosamine dose-dependently increased human VCAM-1 promoter activity. In addition, GFAT overexpression activated the VCAM-1 promoter (2.25-fold), with further augmentation by high glucose and abrogation by inhibitors of GFAT, NF-κB, and O-glycosylation. Inactivation of the two NF-κB sites in the VCAM-1 promoter abolished its response to high glucose, glucosamine, and GFAT overexpression. These results suggest that increased flux through the hexosamine pathway leads to NF-κB–dependent promoter activation in MCs.