The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes

Abstract

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.

Fig. 1

p47^phox-dependent NOX, but not NOX2, mediates diabetic uncoupling of eNOS. WT, p47^phox−/− or Nox2^−/y mice were made diabetic by STZ injection (100 mg/kg per day for 3 days). On day 7, blood glucose levels were determined and aortas were harvested for detection of L-NAME-sensitive superoxide production (reflective of eNOS uncoupling activity) using electron spin resonance. a Blood glucose levels. b Superoxide level inhibited by NOS inhibitor L-NAME (left panel) and iNOS inhibitor 1400W (right panel, c). d eNOS uncoupling activity in p47^phox−/− mice. e eNOS uncoupling activity in Nox2^−/y mice. *p<0.05, **p<0.01, ***p<0.001 vs sham mice; ^††p<0.01 vs STZ-diabetic WT mice (n=9–13)

Fig. 2

NOX1, but not NOX4, mediates diabetic uncoupling of eNOS. C57BL/6 mice were made diabetic by STZ injection (100 mg/kg per day for 3 days) and transfected with control, Nox1 or Nox4 siRNA. On day 7, blood glucose levels were determined and aortas were harvested for L-NAME-sensitive superoxide detection using electron spin resonance. a Protein contents of NOX1 and NOX4 in siRNA-transfected diabetic mouse aortas. b Blood glucose levels. c, d eNOS uncoupling activity in scrambled siRNA, Nox1 or Nox4 siRNA-transfected diabetic mice. *p<0.05, **p<0.01, ***p<0.001 vs sham mice; ^†p<0.05 vs untransfected diabetic mice (n=6–7). White bars, sham mice; black bars, STZ-diabetic mice. RNAi, RNA interference; Sc, scrambled

Fig. 3

NOXO1-dependent NOX1 mediates diabetic uncoupling of eNOS. C57BL/6 and NOX1^−/y mice were made diabetic by STZ injection (100 mg/kg per day for 3 days) and transfected with control or Noxo1 siRNA as described in the Methods section. On day 7, blood glucose levels were determined and aortas were harvested for L-NAME-sensitive superoxide detection using electron spin resonance. a eNOS uncoupling activity in control siRNA or Noxo1 siRNA-transfected diabetic mice. b Protein content of NOXO1 in siRNA-transfected diabetic mouse aortas. c Blood glucose levels. *p<0.05 vs sham mice; ^†p<0.05 vs control siRNA-transfected diabetic mice (n=4–6). White bars, sham mice; black bars, STZ-diabetic mice; RNAi, RNA interference. WT and Nox1^−/y mice were made diabetic by STZ injection (100 mg/kg per day for 3 days). d eNOS uncoupling activity in WT and Nox1^−/y mice. e Vasorelaxation in response to acetylcholine in precontracted aortic rings. f Blood glucose levels. *p<0.05, **p<0.01, ***p<0.001 vs WT mice; ^†††p<0.001 vs Nox1^−/y mice (n=6–7). Black line, STZ-diabetic mice; light grey line, Nox1^−/y/STZ-diabetic mice; medium grey line, Nox1^−/y mice; dark grey line, control mice. g Western blot for NOX1. h Upregulation of NOX1 protein production in STZ-diabetic mice. i quantitative grouped data for NOX1 protein content. **p<0.01 vs WT mice (n=3)

Fig. 4

NOX1-mediated eNOS uncoupling in diabetes is accompanied by deficiencies in H₄B and DHFR. WT and Nox1^−/y mice were made diabetic by STZ injection (100 mg/kg/day for 3 days). On day 7, aortas were harvested for analysis of eNOS level, DHFR level and H₄B content. a Representative western blots for eNOS and DHFR. b Quantified grouped data for eNOS protein content. c Quantified grouped data for DHFR protein content. d HPLC analysis of aortic H₄B content. *p<0.05, **p<0.01 vs WT mice; ^†p<0.05 vs Nox1^−/y mice (n=4)

Fig. 5

Mitochondrion does not contribute to diabetic uncoupling of eNOS. C57BL/6 mice were fed the mitochondrial complex I inhibitor rotenone for 2 days prior to being made diabetic by STZ injection (100 mg/kg per day for 3 days), and throughout the study period of 7 days. Some diabetic mice were transfected with complex III siRNA as described in the Methods section. a Expression of Rieske subunit of mitochondrial complex III. b Circulating levels of rotenone in mice as determined by HPLC analysis (see Methods section). c L-NAME-sensitive superoxide production (eNOS uncoupling activity). d Blood glucose levels. *p<0.05 vs sham mice (n=6). White bars, sham mice; black bars, STZ-diabetic mice; Mito III, mitochondrial complex III; RNAi, RNA interference

Fig. 6

Recoupling of eNOS in diabetes via restoration of DHFR. C57BL/6 mice were made diabetic by STZ injection (100 mg/kg per day for 3 days) and transfected with empty vector or pcDNA3.1-Dhfr plasmid (a–d). Oral administration with folic acid (15 mg/kg per day) was started 2 days prior to induction of diabetes with STZ (100 mg/kg per day for 3 days), and mice were fed with folic acid-containing food throughout the study period of 7 days (e–h). On day 7, blood glucose levels were determined and aortas were harvested for superoxide production using electron spin resonance. a Blood glucose levels. *p<0.05 vs sham. b DHFR levels in diabetic aortas. *p<0.05, ***p<0.001 vs sham. c L-NAME-sensitive superoxide production (uncoupling activity of eNOS). *p<0.05 vs sham mice; ^†p<0.05 vs untransfected diabetic mice. d Vasorelaxation in response to acetylcholine in precontracted aortic rings. **p<0.01, ***p<0.001 vs sham mice; ^††p<0.01, ^†††p<0.001 vs Dhfr-overexpressed sham mice (n=5). Black line, STZ-diabetic mice; light grey line, Dhfr/STZ-diabetic mice; dark grey line, sham mice; medium grey line, Dhfr-overexpressed mice. e L-NAME-sensitive superoxide production (eNOS uncoupling activity). *p<0.05 vs sham mice; ^†p<0.05 vs control diet-treated diabetic mice (n=4). f Blood glucose levels. *p<0.05 vs sham. g Circulating levels of 5-MTHF as determined by HPLC analysis (see Methods section) in mice fed a standard diet or a diet supplemented with folic acid (15 mg/kg per day). h Vasorelaxation in response to acetylcholine in precontracted aortic rings. **p<0.01, ***p<0.001 vs sham mice; ^†p<0.05, ^†††p<0.001 vs folic acid-treated sham mice; ^‡p<0.05 vs folic acid-treated diabetic mice (n=4–6). Black line, STZ-diabetic mice; medium grey line, folic acid-treated STZ diabetic mice; light grey line, folic acid diet-fed mice; dark grey line, WT mice; white bars, sham mice; black bars, STZ-diabetic mice

Fig. 7

Proposed mechanisms of action for an intermediate role of p47phox and NOXO1-dependent NOX1 activation in diabetic uncoupling of eNOS. NOXA1, NOX activator 1