Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 97, Issue 22, Pages 12222-12226Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.97.22.12222
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- NIDDK NIH HHS [DK-33861, R01 DK033861, R56 DK033861] Funding Source: Medline
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The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Spl followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta (1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Spl sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Spl sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Spl, which activates expression of genes that contribute to the pathogenesis of diabetic complications.
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