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-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.

Figure 1

Schematic mechanism by which hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and increases Sp1-dependent gene expression. Increased glycolysis generates superoxide by increasing the proton electrochemical gradient generated by the mitochondrial electron transport chain (1, 34). Superoxide, thus generated, partially inhibits GAPDH enzymatic activity, diverting fructose-6-phosphate into the hexosamine pathway and increasing UDP-GlcNAc. This increase leads to increased O-GlcNAcylation of Sp1, increased SP1 transactivation, and Sp1-dependent gene expression.

Figure 2

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction and of a hexosamine pathway inhibitor on GAPDH activity. AZA, azaserine. *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cells incubated in 30 mM glucose. n = 6.

Figure 3

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction and azaserine (AZA) on hexosamine pathway activity. *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cells incubated in 30 mM glucose. n = 3.

Figure 4

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction and azaserine (AZA) on Sp1 O-linked GlcNAc, phosphoserine, and phosphothreonine. (A) Representative IP-Western blot. (B) Sp1 O-linked GlcNAc (relative densitometric means of three IP-Western blots). (C) Sp1 phosphoserine (relative densitometric means of three IP-Western blots). (D) Sp1 phosphothreonine (relative densitometric means of three IP-Western blots). *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cell incubated in 30 mM glucose. n = 3.

Figure 5

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction by UCP-1 and MnSOD on Sp1 O-linked GlcNAc, phosphoserine, and phosphothreonine. (A) Representative IP-Western blot. (B) Sp1 O-linked GlcNAc (relative densitometric means of three IP-Western blots). (C) Sp1 phosphoserine (relative densitometric means of three IP-Western blots). (D) Sp1 phosphothreonine (relative densitometric means of three IP-Western blots). *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cells incubated in 30 mM glucose. n = 3.

Figure 6

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction and azaserine (aza) on TGFβ₁ (A) and PAI-1 (B) promoter activity. *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cells incubated in 30 mM glucose. n = 3.

Figure 7

Effect of inhibitors of hyperglycemia-induced mitochondrial superoxide overproduction and azaserine on PAI-1 promoter activity from a truncated promoter containing either two Sp1 binding sites (pGL 85) or two mutated Sp1 binding sites (pGL 85 mSP-1_{A + B}). *, P < 0.01 compared to cells incubated in 5 mM glucose. #, P < 0.01 compared to cells incubated in 30 mM glucose. n = 3.