Xue-Liang Du1, Diane Edelstein1, Luciano Rossetti1, I. George Fantus1, Harry Goldberg1, Fuad N. Ziyadeh1, Jie Wu1, Michael Brownlee1
1Albert Einstein College of Medicine, Diabetes Research Center,
Bronx, NY 10461; Department of Medicine, Mount Sinai
Hospital, Toronto, ON, M5G 1X5 Canada; and Department of
Medicine, Renal-Electrolyte, and Hypertension Division, University of
Pennsylvania, Philadelphia, PA 19104-6144
Tóm tắt
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-β
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.
