Differential gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients with Type 2 diabetes and microangiopathy.
OBJECTIVE
While the downstream effects of increased reactive oxygen species (ROS) in the pathogenesis of diabetes were well studied, only a few studies have explored the cellular sources of ROS. We examined whether protection against oxidative stress is altered in patients with diabetes and microangiopathy by examining changes in NADPH oxidase (p22(phox)) and hemoxygenase-1 (HO-1) levels.


METHODS
NADPH oxidase (p22(phox)) and HO-1 gene expression were probed by RT-PCR using leucocytes from patients with Type 2 diabetes without (n = 19) and with microangiopathy (n = 20) and non-diabetic subjects (n = 17). Levels of lipid peroxidation as measured by thiobarbituric reactive substances (TBARS) and protein carbonyl content (PCO) were determined by fluorimetric and spectrophotometric methods, respectively.


RESULTS
p22(phox) gene expression (mean +/- SE) was significantly (P < 0.05) higher in diabetic patients with (0.99 +/- 0.04) and without microangiopathy (0.86 +/- 0.05) compared with control subjects (0.66 +/- 0.05). Consistent with the mRNA data, the p22(phox) protein expression and NADPH oxidase activity was also increased in cells from diabetic patients compared with control subjects. However, HO-1 gene expression was significantly (P < 0.05) lower in patients with (0.73 +/- 0.03) and without microangiopathy (0.85 +/- 0.02) compared with control subjects (1.06 +/- 0.03). The mean (+/- SE) levels of TBARS were significantly (P < 0.05) higher in diabetic patients with (14.36 +/- 1.3 nM/ml) and without microangiopathy (12.20 +/- 1.3 nM/ml) compared with control subjects (8.58 +/- 0.7 nM/ml). The protein carbonyl content was also significantly (P < 0.05) higher in diabetic patients with (1.02 +/- 0.04 nmol/mg protein) and without microangiopathy (0.84 +/- 0.06 nmol/mg protein) compared with control subjects (0.48 +/- 0.02 nmol/mg protein). In diabetic subjects, increased p22(phox) gene expression was negatively correlated with HO-1 and positively correlated with TBARS, PCO, HbA(1c) and diabetes duration. In contrast, HO-1 gene expression was correlated negatively with p22phox, TBARS, PCO, HbA(1c) and diabetes duration.


CONCLUSIONS
Our results indicate that increased oxidative damage is seen in Asian Indians with Type 2 diabetes and microangiopathy and is associated with increased NADPH oxidase (p22(phox)) and decreased HO-1 gene expression.
Adaikalakoteswari
A
A
Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, Gopalapuram, Chennai, India.
Balasubramanyam
M
M
Rema
M
M
Mohan
V
V
D044466:Asian People; D016022:Case-Control Studies; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology* / Q000208:ethnology; D003925:Diabetic Angiopathies / Q000201:enzymology* / Q000208:ethnology; D015870:Gene Expression; D006442:Glycated Hemoglobin / Q000032:analysis; D051547:Heme Oxygenase-1 / Q000235:genetics*; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D015227:Lipid Peroxidation; D019255:NADPH Oxidases / Q000235:genetics*; D010084:Oxidation-Reduction; D050050:Protein Carbonylation; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis; D013997:Time Factors
D006442:Glycated Hemoglobin; D051547:Heme Oxygenase-1; D019255:NADPH Oxidases; D012333:RNA, Messenger; D017392:Thiobarbituric Acid Reactive Substances; C086986:CYBA protein, human