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. 2012 Jul;61(7):1838-47.
doi: 10.2337/db11-1241. Epub 2012 May 14.

Uncoupling endothelial nitric oxide synthase is ameliorated by green tea in experimental diabetes by re-establishing tetrahydrobiopterin levels

Aline M Faria  1 , Alexandros Papadimitriou, Kamila C Silva, Jacqueline M Lopes de Faria, José B Lopes de Faria
Affiliations

Uncoupling endothelial nitric oxide synthase is ameliorated by green tea in experimental diabetes by re-establishing tetrahydrobiopterin levels

Aline M Faria et al. Diabetes. 2012 Jul.

Abstract

The current study investigated the potential of green tea (GT) to improve uncoupling of endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes, nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in tetrahydrobiopterin (BH(4)) levels and a decrease in the eNOS dimer-to-monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH(4) and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and was normalized by GT. Exogenous administration of BH(4) in ihMCs reversed the HG-induced rise in ROS and the decline in NO production. However, coadministration of GT with BH(4) did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes-induced reduction of BH(4) levels, ameliorating uncoupling eNOS, and thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy.

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Figures

FIG. 1.
FIG. 1.
A: Nitrite (NO2−) and nitrate (NO3−), the stable NO end products, were quantified as a measurement of NO levels in renal cortical homogenates by Griess reaction in SHR rats: control (CT), diabetic (DM), and DM treated with GT (DM GT). Results were corrected for the protein concentration and are expressed as NOx− (μmol/mg of protein). *P = 0.02 vs. SHR CT; †P = 0.05 vs. SHR DM. B: Representative Western blots of the renal cortical eNOS and p-eNOS (Thr495) expression from SHR CT rats, SHR DM rats, and SHR DM GT. Densitometric analysis of the eNOS–to–β-actin ratio (C), phosphorylated (p)-eNOS (Thr495)-to-β-actin ratio (*P = 0.002 vs. SHR CT, †P = 0.005 vs. SHR DM) (D), and p-eNOS (Ser1177)–to–β-actin ratio (*P = 0.03 vs. SHR CT, †P = 0.05 vs. SHR DM) (E), in the three SHR rat groups. Bars represent means ± SD. F: Representative Western blots of the renal cortical of eNOS dimer and monomer expression from SHR CT rats, SHR DM rats, and SHR DM GT rats. G: Densitometric analysis of the percentage of eNOS dimer-to-monomer ratio. *P = 0.02 vs. SHR CT. †P = 0.05 vs. SHR DM.
FIG. 2.
FIG. 2.
Ultraperformance liquid chromatography was used to analyze total biopterin and BH4 expression in SHR control (CT) rats, diabetic (DM) rats, and DM rats fed GT (DM GT) in the renal cortex (A) and urine (B). Results in the renal cortex were corrected for the protein concentration and are expressed as nmol/mg protein. *P = 0.002 vs. SHR CT. †P < 0.0001 vs. SHR DM. ‡P < 0.0001 vs. SHR CT. Results in the urine were corrected for 24-h urine volume. *P < 0.0001 vs. SHR CT. Representative graphs show the of percentage of BH4 to BH2 oxidation in the renal cortex (C) (*P = 0.01 vs. SHR CT, †P = 0.03 vs. SHR DM) and in the urine (D) (*P = 0.004 vs. SHR CT, †P = 0.05 vs. SHR DM).
FIG. 3.
FIG. 3.
A: Representative photomicrographs of diaminorhodamine-4M AM indicating NO production. ihMCs were cultured for 24 h in NG (5.5 mmol/L), HG (30 mmol/L), and HG with GT (100 µg/mL). B: Quantification of NO levels in ihMCs via DAF-2DA. Values are mean ± SD and expressed as the percentage of fluorescence. Values were corrected by the number of cells at the end of each treatment. *P = 0.03 vs. NG. †P = 0.02 vs. HG. C: Representative Western blots of the ihMCs of eNOS expression from ihMCs cultured under NG, HG, and HG treated with GT for 24 h. D: Densitometric analysis of the eNOS–to–β-actin ratio. E: Representative Western blots of the ihMCs of eNOS dimer and monomer expression from ihMCs cultured under NG, HG, and HG treated with GT for 24 h. F: Densitometric analysis of the percentage of eNOS dimer-to-monomer ratio.*P = 0.04 vs. NG. †P = 0.05 vs. HG. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
A: Representative photomicrographs of ihMCs probe with H2DCF-DA indicating ROS production. Cells were cultured in NG, HG, HG plus GT, HG plus DPI, and HG plus L-NAME. B: Quantification of total intracellular ROS levels and an assessment of enzymatic sources of ROS production in ihMCs. HMCs were cultured for 24 h in NG (5.5 mmol/L) and HG (30 mmol/L) in the presence and absence of GT. Mannitol was used as an osmotic control; L-NAME (an inhibitor of NOS), DPI (an inhibitor of NADPH oxidase), and rotenone (an inhibitor of mitochondrial complex I) were also used at HG levels to define the enzymatic sources of ROS production. Values are mean ± SD and expressed as the percentage of fluorescence. Values were corrected for the number of cells at the end of each treatment. *P < 0.0001 vs. NG. †P = 0.04 vs. HG. ‡P = 0.03 vs. HG. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
A: Total biopterin and BH4 expression analysis by ultraperformance liquid chromatography in ihMC lysate. Results were corrected for the protein concentration and expressed as nmol/mg protein. *P = 0.05 vs. NG. †P = 0.02 vs. HG. ‡P = 0.04 vs. HG. B: Representative graphs of percentage of BH4 to BH2 oxidation in ihMC lysate. *P = 0.01 vs. NG. †P = 0.01 vs. HG. C: Quantification of total intracellular ROS levels by H2DCF-DA via a fluorimeter after incubation of ihMCs for 24 h in NG and HG mediums in the presence also of BH4 (1, 10, and 100 μmol/L). *P = 0.0001 vs. NG. †P = 0.004 vs. HG. D: NO levels were also quantified after incubation with DAF-2DA via a fluorimeter. *P = 0.007 vs. NG. †P = 0.001 vs. HG. The bars represent mean ± SD. Values are expressed as the percentage of fluorescence units and were corrected by the number of cells at the end of each treatment.
FIG. 6.
FIG. 6.
A: GTPCH I activity was measured in HPLC by the concentration of neopterin in cell lysate. Results were corrected for the protein concentration and expressed as the percentage of pmol/μg protein. The levels of neopterin indicate GTPCH activity. The ihMCs were cultured for 24 h in NG (5.5 mmol/L), HG (30 mmol/L), and HG with GT (100 µg/mL). *P = 0.03 vs. NG. †P = 0.009 vs. HG. B: Representative photomicrographs of H2DCF-DA in ihMCs indicating ROS production. ihMCs were kept for 24 h in NG and in HG medium in the presence also of BH2 (10 μmol/L). C: ROS measurement in ihMCs supplemented with BH2 (1, 10, and 100 μmol/L). *P = 0.0001 vs. NG. †P = 0.03 vs. HG. D: Quantitative analysis of intracellular NO levels were also carried out after incubation with DAF-2DA. *P = 0.007 vs. NG. †P = 0.001 vs. HG. ROS measurement in ihMCs supplemented with BH2 (1, 10, and 100 μmol/L). *P = 0.0001 vs. NG. †P = 0.03 vs. HG. E: ROS measurement in ihMCs pretreated with HG and GT and supplemented with 2,4-diamino-6-hydroxypyrimidine (DAHP; 100 μmol/L, 500 μmol/L, and 1 mmol/L). *P = 0.0001 vs. NG. †P = 0.0001 vs. HG. ‡P = 0.0003 vs. HG. F: Quantitative analysis of intracellular NO levels in ihMCs pretreated with HG and GT and supplemented with DAHP. *P = 0.0004 vs. NG. †P = 0.0001 vs. HG. ‡P = 0.002 vs. NG. The bars represent mean ± SD. Values are expressed as the percentage of fluorescence units and were corrected for the number of cells at the end of each treatment. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 7.
FIG. 7.
A: Representative photomicrographs of H2DCF-DA in ihMCs indicating ROS production. ihMCs were kept for 24 h in NG and HG mediums in the presence also of GT (100 μg/mL). B: Total intracellular ROS levels by H2DCF-DA was quantified via a fluorimeter. *P = 0.0001 vs. NG. †P = 0.0001 vs. HG. C: ihMCs were cultured in HG and supplemented with GT (100 µg/mL) and BH4, followed by measurement of intracellular ROS by H2DCF-DA. *P < 0.0001 vs. NG. †P < 0.0001 vs. HG. The bars represent mean ± SD. Values are expressed as the percentage of fluorescence units. (A high-quality digital representation of this figure is available in the online issue.)
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