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transcription is not certain, but the observation that binding sites for
the transcription factor Sp1 regulate hyperglycaemia-induced 
activation of the PAI-1 promoter in vascular smooth muscle cells
77
suggested that covalent modification of Sp1 by N-acetylglucosamine
(GlcNAc) might explain the link between activation of the 
hexosamine pathway and hyperglycaemia-induced changes in 
transcription of the gene for PAI-1. Glucosamine itself was 
subsequently shown to activate the PAI-1 promoter through Sp1 
sites in glomerular mesangial cells
78
. The glycosylated form of 
Sp1 seems to be more transcriptionally active than the deglycosylated
form
79
. A fourfold increase in O-acetylglucosaminylation of Sp1
caused by inhibition of the enzyme O-GlcNAc-
b-N-acetylglu-
cosaminidase resulted in a reciprocal 30% decrease in the level of 
serine–threonine phosphorylation of Sp1, supporting the concept
that O-acetylglucosaminylation and phosphorylation compete for
the same sites on this protein
80
.
Recently, hyperglycaemia was shown to induce a 2.4-fold increase
in hexosamine pathway activity in aortic endothelial cells, resulting
in a 1.7-fold increase in Sp1 O-linked GlcNAc and a 70–80% decrease
in Sp1 O-linked phosphothreonine and phosphoserine
10
. Concomi-
tantly, hyperglycaemia resulted in a 3.8-fold increase in expression
from an 85-base-pair truncated PAI-1 promoter–luciferase reporter
DNA containing two Sp1 sites, but failed to increase expression when
the two Sp1 sites were mutated
10
.
Modification of Sp1 by GlcNAc may regulate other glucose-
responsive genes in addition to that for PAI-1. As virtually every RNA
polymerase II transcription factor examined has been found to be O-
acetylglucosaminylated
81
, it is possible that reciprocal modification
by O-acetylglucosaminylation and phosphorylation of transcription
factors other than Sp1 may function as a more generalized 
mechanism for regulating glucose-responsive gene transcription.
In addition to transcription factors, many other nuclear and cyto-
plasmic proteins are dynamically modified by O-linked GlcNAc, and
may show reciprocal modification by phosphorylation in a manner
analogous to Sp1 (ref. 81). One example relevant to diabetic compli-
cations is the inhibition of eNOS activity by hyperglycaemia-induced
O-acetylglucosaminylation at the Akt site of the eNOS protein
82
.
Other examples might be various PKC isoforms, which are activated
by glucosamine without membrane translocation (H. J. Goldberg, 
C. J. Whiteside & G. Fantus, personal communication).
Thus, activation of the hexosamine pathway by hyperglycaemia
may result in many changes in both gene expression and protein
function, which together contribute to the pathogenesis of diabetic
complications.

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