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

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-␤ 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. D iabetic hyperglycemia causes a variety of pathologic changes in small vessels, arteries, and peripheral nerves (1, 2). Three major hypotheses about how hyperglycemia causes diabetic complications have generated extensive data as well as several clinical trials based on specific inhibitors of these pathways (3-6). These three pathways-activation of protein kinase C isoforms (7), increased formation of glucose-derived advanced glycation endproducts (3), and increased glucose flux through the aldose reductase pathway (8)-recently have been shown to be consequences of a single common mechanism, hyperglycemia-induced mitochondrial superoxide overproduction (1).A fourth hypothesis about how hyperglycemia causes diabetic complications has been formulated recently (9, 10), in which glucose is shunted into the hexosamine pathway. Inhibition of the rate-limiting enzyme in the conversion of glucose to glucosamine, glutamine:fructose-6-phosphate amidotransferase, bl...

Phenotype-specific inhibition of the vascular smooth muscle cell cycle by high glucose treatment

“…Although activation of PKC by glucose treatment could be mediated by the production of diacylglycerol, our data showing that glucosamine mimics the inhibitory effect of glucose treatment on SMC proliferation and that inhibition of glutamine:fructose-6-phosphate aminotransferase reverses this effect strongly suggest involvement of the hexosamine pathway in activation of PKC and the effects of glucose treatment on SMCs. In addition, the increase in ROS production induced by glucose treatment was reported to promote the formation of glucosamine through the hexosamine pathway [43]. Therefore, our data showing that glucose treatment increased the production of ROS in epithelioid SMCs further support the involvement of the hexosamine pathway.…”

Regulation of human resistin gene expression in cell systems: an important role of stimulatory protein 1 interaction with a common promoter polymorphic site

“…O-glycosylation of Sp1 affects the nuclear localisation, stability and transcriptional activity of Sp1 and phosphorylation regulates the binding activity of Sp1 to the target sequence [22]. It has been reported that hyperglycaemia increases glycosylation of Sp1 and influences the transcriptional activity of Sp1 [37]. Since resistin expression is increased by hyperglycaemia in rodents, it will be important to find the mechanism by which hyperglycaemia regulates resistin expression.…”

Mechanisms by which diabetes increases cardiovascular disease

“…Hyperglycemia-induced activity of GFAT has been associated with transcription of transforming growth facto-β1 (TGF-β1) and plasminogen activator inhibitor-1 (PAI-1) in bovine aortic endothelial cells (BAEC) [31]. This effect was inhibited by treatment with azaserine, a GFAT inhibitor [31].…”

“…Hyperglycemia-induced activity of GFAT has been associated with transcription of transforming growth facto-β1 (TGF-β1) and plasminogen activator inhibitor-1 (PAI-1) in bovine aortic endothelial cells (BAEC) [31]. This effect was inhibited by treatment with azaserine, a GFAT inhibitor [31]. Similarly, inhibition of GFAT by antisense oligonucleotides resulted in reversal of hyperglycemia-induced inhibition of eNOS activity in BAEC, a finding that was confirmed in vivo in aortas from rats with STZ-induced diabetes [32].…”