Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation

Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):12222-6. doi: 10.1073/pnas.97.22.12222.

Abstract

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 Sp1 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 Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosamine / metabolism
  • Animals
  • Cattle
  • Cells, Cultured
  • Glycosylation
  • Hexosamines / metabolism*
  • Hyperglycemia / metabolism*
  • Mitochondria / metabolism*
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Plasminogen Activator Inhibitor 1 / genetics
  • Plasminogen Activator Inhibitor 1 / metabolism*
  • Promoter Regions, Genetic
  • Sp1 Transcription Factor / metabolism*
  • Superoxides / metabolism*
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism

Substances

  • Hexosamines
  • Plasminogen Activator Inhibitor 1
  • Sp1 Transcription Factor
  • Transforming Growth Factor beta
  • Superoxides
  • Phosphothreonine
  • Phosphoserine
  • Acetylglucosamine