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

Xue-Liang Du, Diane Edelstein, Luciano Rossetti, Ivan George Fantus, Howard Goldberg, Fuad Ziyadeh, Jie Wu, Michael Brownlee

Research output: Contribution to journalArticle

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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 I 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-β1 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.

Original languageEnglish (US)
Pages (from-to)12222-12226
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number22
DOIs
StatePublished - Oct 24 2000

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Hexosamines
Plasminogen Activator Inhibitor 1
Glycosylation
Superoxides
Hyperglycemia
Luciferases
Phosphothreonine
Phosphoserine
Glyceraldehyde-3-Phosphate Dehydrogenases
Diabetes Complications
Superoxide Dismutase
Thenoyltrifluoroacetone
Electron Transport Complex II
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
Azaserine
Glucosamine
Oxidative Phosphorylation
Transforming Growth Factors
Reporter Genes
Immunoprecipitation

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. / Du, Xue-Liang; Edelstein, Diane; Rossetti, Luciano; Fantus, Ivan George; Goldberg, Howard; Ziyadeh, Fuad; Wu, Jie; Brownlee, Michael.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 22, 24.10.2000, p. 12222-12226.

Research output: Contribution to journalArticle

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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 I 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-β1 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.",
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AU - Ziyadeh, Fuad

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