AIMS/HYPOTHESIS: We have previously shown that NADPH oxidase (NOX) lies upstream 
of uncoupled endothelial nitric oxide synthase (eNOS), which is known to occur 
in diabetic endothelium. However, it remains unclear which specific NOX 
isoform(s) is responsible for eNOS uncoupling and endothelial dysfunction in 
diabetic mouse models. The aim of the present study was to test the hypothesis 
that one or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS, which 
has been shown to occur in patients with diabetes to contribute to endothelial 
dysfunction.
METHODS: Diabetes was induced by streptozotocin administration. The N 
(ω)-nitro-L-arginine methyl ester (L-NAME)-sensitive superoxide production of 
aortic segments, reflective of eNOS uncoupling activity, was determined by 
electron spin resonance.
RESULTS: The L-NAME-sensitive superoxide production was more than doubled in 
wild-type diabetic mice, implicating uncoupling of eNOS. This was abolished in 
diabetic p47 ( phox-/-) (also known as Ncf1 (-/-)) mice, but preserved in Nox2 
(-/y) (also known as Cybb (-/-)) mice made diabetic. The eNOS uncoupling 
activity was markedly attenuated in diabetic mice transfected with Nox1 or Nox1 
organiser 1 (Noxo1) short interfering RNA (siRNA), and abolished in Nox1 (-/y) 
diabetic mice. Diabetes-induced impairment in endothelium-dependent 
vasorelaxation was also significantly attenuated in the Nox1 (-/y) mice made 
diabetic. By contrast, Nox4 siRNA, or inhibition of mitochondrial complex I or 
III with rotenone or siRNA, respectively, had no effect on diabetic uncoupling 
of eNOS. Overexpression of Dhfr, or oral administration of folic acid to improve 
dihydrofolate reductase (DHFR) function, recoupled eNOS in diabetes to improve 
endothelial function.
CONCLUSIONS/INTERPRETATION: Our data demonstrate for the first time that the 
p47(phox) and NOXO1-dependent activation of NOX1, but not that of NOX2, NOX4 or 
mitochondrion, mediates diabetic uncoupling of eNOS. NOX1-null mice are 
protected from diabetic endothelial dysfunction. Novel approaches to inhibit 
NOX1 and/or improve DHFR function, may prove to have therapeutic potential for 
diabetic endothelial dysfunction.