title: Flux through the hexosamine pathway is a determinant of nuclear factor kappaB- 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 kappaB (NF-kappaB)-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-kappaB 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-kappaB enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 micromol/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-kappaB consensus sequences. Immunoblotting revealed that the p65 subunit of NF-kappaB 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-kappaB, and O-glycosylation. Inactivation of the two NF-kappaB 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-kappaB-dependent promoter activation in MCs.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada. leighton_james@med.unc.edu
authors.forename: Leighton R
authors.initials: LR
authors.lastname: James
authors.forename: Damu
authors.initials: D
authors.lastname: Tang
authors.forename: Alistair
authors.initials: A
authors.lastname: Ingram
authors.forename: Hao
authors.initials: H
authors.lastname: Ly
authors.forename: Kerri
authors.initials: K
authors.lastname: Thai
authors.forename: Lu
authors.initials: L
authors.lastname: Cai
authors.forename: James W
authors.initials: JW
authors.lastname: Scholey
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001783:Blood Flow Velocity / Q000187:drug effects; D001786:Blood Glucose / Q000187:drug effects / Q000378:metabolism; D001794:Blood Pressure / Q000187:drug effects; D002196:Capillaries / Q000187:drug effects / Q000502:physiology*; D005217:Fat Emulsions, Intravenous / Q000008:administration & dosage / Q000494:pharmacology; D005263:Femoral Artery / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D015309:Glucose Clamp Technique; D006339:Heart Rate / Q000187:drug effects; D006439:Hemodynamics / Q000187:drug effects / Q000502:physiology*; D006946:Hyperinsulinism; D007262:Infusions, Intravenous; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012044:Regression Analysis; D014280:Triglycerides / Q000008:administration & dosage / Q000494:pharmacology*; D014655:Vascular Resistance / Q000187:drug effects
chemical_list: D001786:Blood Glucose; D005217:Fat Emulsions, Intravenous; D007328:Insulin; D014280:Triglycerides; D005947:Glucose