PMC

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

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)

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)

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

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

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)

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