Du X L, Edelstein D, Rossetti L, Fantus I G, Goldberg H, Ziyadeh F, Wu J, Brownlee M
Albert Einstein College of Medicine, Diabetes Research Center, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):12222-6. doi: 10.1073/pnas.97.22.12222.
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.
己糖胺途径与糖尿病并发症的发病机制有关。我们首先确定，高血糖通过增加线粒体超氧化物的产生以及使己糖胺途径活性伴随增加2.4倍，导致牛主动脉内皮细胞中甘油醛-3-磷酸脱氢酶活性降低。电子传递复合物II的抑制剂（噻吩甲酰三氟丙酮）、氧化磷酸化解偶联剂（羰基氰化物间氯苯腙）、超氧化物歧化酶模拟物[锰（III）四（4-苯甲酸）卟啉]、解偶联蛋白1或锰超氧化物歧化酶的过表达以及己糖胺途径限速酶（谷氨酰胺：果糖-6-磷酸酰胺转移酶）的抑制剂氮丝氨酸，均可完全阻止甘油醛-3-磷酸脱氢酶活性降低和己糖胺途径活性增加。用抗O-连接的N-乙酰葡糖胺、磷酸丝氨酸和磷酸苏氨酸的抗体进行蛋白质免疫沉淀后再进行蛋白质印迹分析表明，高血糖使N-乙酰葡糖胺增加1.7倍，磷酸丝氨酸减少80%，磷酸苏氨酸减少70%。相同的抑制剂可阻止所有这些变化。高血糖使转化生长因子-β1启动子荧光素酶报告基因的表达增加2倍，并使（-740至+44）纤溶酶原激活物抑制剂-1启动子荧光素酶报告基因的表达增加近3倍。抑制线粒体超氧化物的产生或葡糖胺途径可阻止所有这些变化。高血糖以类似方式（3.8倍）使含有两个Sp1位点的85 bp截短型纤溶酶原激活物抑制剂-1（PAI-1）启动子荧光素酶报告基因的表达增加。相比之下，当两个Sp1位点发生突变时，高血糖则无影响。因此，高血糖诱导的线粒体超氧化物过量产生增加了己糖胺的合成以及Sp1的O-糖基化，从而激活了有助于糖尿病并发症发病机制的基因表达。
