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. 2008 Oct;57(10):2626-36.
doi: 10.2337/db07-1579. Epub 2008 Jul 3.

Mechanism of oxidative DNA damage in diabetes: tuberin inactivation and downregulation of DNA repair enzyme 8-oxo-7,8-dihydro-2'-deoxyguanosine-DNA glycosylase

Simona Simone  1 , Yves Gorin, Chakradhar Velagapudi, Hanna E Abboud, Samy L Habib
Affiliations

Mechanism of oxidative DNA damage in diabetes: tuberin inactivation and downregulation of DNA repair enzyme 8-oxo-7,8-dihydro-2'-deoxyguanosine-DNA glycosylase

Simona Simone et al. Diabetes. 2008 Oct.

Abstract

Objective: To investigate potential mechanisms of oxidative DNA damage in a rat model of type 1 diabetes and in murine proximal tubular epithelial cells and primary culture of rat proximal tubular epithelial cells.

Research design and methods: Phosphorylation of Akt and tuberin, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels, and 8-oxoG-DNA glycosylase (OGG1) expression were measured in kidney cortical tissue of control and type 1 diabetic animals and in proximal tubular cells incubated with normal or high glucose.

Results: In the renal cortex of diabetic rats, the increase in Akt phosphorylation is associated with enhanced phosphorylation of tuberin, decreased OGG1 protein expression, and 8-oxodG accumulation. Exposure of proximal tubular epithelial cells to high glucose causes a rapid increase in reactive oxygen species (ROS) generation that correlates with the increase in Akt and tuberin phosphorylation. High glucose also resulted in downregulation of OGG1 protein expression, paralleling its effect on Akt and tuberin. Inhibition of phosphatidylinositol 3-kinase/Akt significantly reduced high glucose-induced tuberin phosphorylation and restored OGG1 expression. Hydrogen peroxide stimulates Akt and tuberin phosphorylation and decreases OGG1 protein expression. The antioxidant N-acetylcysteine significantly inhibited ROS generation, Akt/protein kinase B, and tuberin phosphorylation and resulted in deceased 8-oxodG accumulation and upregulation of OGG1 protein expression.

Conclusions: Hyperglycemia in type 1 diabetes and treatment of proximal tubular epithelial cells with high glucose leads to phosphorylation/inactivation of tuberin and downregulation of OGG1 via a redox-dependent activation of Akt in renal tubular epithelial cells. This signaling cascade provides a mechanism of oxidative stress-mediated DNA damage in diabetes.

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Figures

FIG. 1.
FIG. 1.
Diabetes increases Akt and tuberin phosphorylation and decreases OGG1 protein expression. A–C: Representative immunoblot shows an increase in phospho-Akt (p-Akt) (A) and phospho-tuberin (p-tuberin) (B) and a decrease in OGG1 expression (C) in homogenized kidney cortex of diabetic (D) compared with control (C) rats. GAPDH was used as a loading control.
FIG. 2.
FIG. 2.
Diabetes causes a decrease in OGG1 protein expression and increases 8-oxodG levels. A: Kidney cortex sections were stained with anti-OGG1 antibody. There is a decrease in OGG1 staining in kidney cortex of diabetic animals compared with the controls. B: 8-OxodG levels are higher in mitochondrial DNA fraction of kidney cortex of diabetic rats compared with control rats. Data are expressed as picomoles 8-oxodG/dG × 10−5 in 90 μl DNA hydrolysate. Significant difference from wild-type cells is indicated by **P < 0.01. (Please see http://dx.doi.org/10.2337/db07-1579 for a high-quality digital representation of this image.)
FIG. 3.
FIG. 3.
Effects of high glucose (HG) on Akt/PKB and tuberin phosphorylation and OGG1 expression in MCT cells. A–C: Representative immunoblot shows an increase in phospho-Akt (p-Akt) (A) and phospho-tuberin (p-tuberin) (B) in MCT cells treated with high glucose (25 mmol/l glucose d-glucose) for the time periods indicated. GAPDH was used as loading control. Histograms in the bottom panel represent means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. D: Immunoflourescence shows an increase in mitochondrial and nuclear 8-oxodG staining in MCT treated with high glucose for 1 h—an effect that was more intense at 2 and 4 h compared with normal glucose. FITC for 8-oxodG (green color) and propidium iodide for nuclear staining (red color) were detected with excitation wavelengths at 450–490 nm and 535 nm, respectively. NG, normal glucose. (Please see http://dx.doi.org/10.2337/db07-1579 for a high-quality digital representation of this figure.)
FIG. 4.
FIG. 4.
Role of PI 3-kinase activation by high glucose (HG) on OGG1 expression in MCT cells. A and C: Representative immunoblot shows a decrease in phospho-Akt (p-Akt) expression in cells preincubated with 50 μmol/l LY294002 and 100 nmol/l Wortmannin, respectively, before exposure to high glucose for 60 min. B and D: Representative immunoblot shows an increase in OGG1 expression in cells preincubated with 50 μmol/l LY294002 and 100 nmol/l Wortmannin before exposure to high glucose for 60 min. Histograms in the bottom panel represent means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. NG, normal glucose.
FIG. 5.
FIG. 5.
Role of Akt/PKB phosphorylation by high glucose (HG) on OGG1 expression in MCT cells. A: Representative immunoblot shows a decrease in phospho-Akt (p-Akt) and phospho-tuberin (A) and an increase in OGG1 expression (B) in cells preincubated with 25 μmol/l Akt inhibitor IV before exposure to high glucose for 60 min. Histograms in the bottom panel represent means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. NG, normal glucose.
FIG. 6.
FIG. 6.
Effects of hydrogen peroxide on tuberin and Akt/PKb phosphorylation and OGG1 expression in MCT cells. A–C: Representative immunoblot shows an increase in phospho-Akt (p-Akt) (A) and phospho-tuberin (p-tuberin) (B) and a decrease in OGG1 expression (C) in MCT cells treated with 100 μmol/l H2O2. GAPDH was used as loading control. Histograms in the bottom panel represent means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. HG, high glucose; NG, normal glucose.
FIG. 7.
FIG. 7.
Effect of high glucose (HG) concentration on the production of ROS in MCT cells. A: DCF fluorescence reflecting the relative levels of ROS was imaged with a confocal laser scanning fluorescence microscope in serum-deprived MCT cells treated with high glucose for the indicated time periods. B: DCF fluorescence was measured using the peroxide-sensitive fluorescent probe DCF-DA by a multiwell fluorescence plate reader in intact MCT cells treated with high glucose. Histogram represents means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. C: NAC blocks ROS generation measured using the peroxide-sensitive fluorescent probe DCF-DA in MCT cells treated with high glucose. Histogram represents means ± SE of three independent experiments. Significant difference from normal glucose (NG) is indicated by **P < 0.01 and from cells treated with NAC versus high glucose alone is indicated by ††P < 0.01. D: Effect of NAC on high glucose–induced Akt/PKB and tuberin phosphorylation and OGG1 expression in MCT cells. Representative immunoblot shows a decrease in phospho-Akt (p-Akt) and phospho-tuberin (p-tuberin) and an increase in OGG1 expression in cells preincubated with NAC before exposure to high glucose. E: NAC blocks 8-oxodG generation in cells treated with high glucose. Immunoflourescence shows a decrease in mitochondrial and nuclear 8-oxodG staining in MCT pretreated with NAC compared with cells treated with high glucose alone for 2 h. (Please see http://dx.doi.org/10.2337/db07-1579 for a high-quality digital representation of this image.)
FIG. 7.
FIG. 7.
Effect of high glucose (HG) concentration on the production of ROS in MCT cells. A: DCF fluorescence reflecting the relative levels of ROS was imaged with a confocal laser scanning fluorescence microscope in serum-deprived MCT cells treated with high glucose for the indicated time periods. B: DCF fluorescence was measured using the peroxide-sensitive fluorescent probe DCF-DA by a multiwell fluorescence plate reader in intact MCT cells treated with high glucose. Histogram represents means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01. C: NAC blocks ROS generation measured using the peroxide-sensitive fluorescent probe DCF-DA in MCT cells treated with high glucose. Histogram represents means ± SE of three independent experiments. Significant difference from normal glucose (NG) is indicated by **P < 0.01 and from cells treated with NAC versus high glucose alone is indicated by ††P < 0.01. D: Effect of NAC on high glucose–induced Akt/PKB and tuberin phosphorylation and OGG1 expression in MCT cells. Representative immunoblot shows a decrease in phospho-Akt (p-Akt) and phospho-tuberin (p-tuberin) and an increase in OGG1 expression in cells preincubated with NAC before exposure to high glucose. E: NAC blocks 8-oxodG generation in cells treated with high glucose. Immunoflourescence shows a decrease in mitochondrial and nuclear 8-oxodG staining in MCT pretreated with NAC compared with cells treated with high glucose alone for 2 h. (Please see http://dx.doi.org/10.2337/db07-1579 for a high-quality digital representation of this image.)
FIG. 8.
FIG. 8.
Effect of high glucose (HG) concentration on Akt/PKB and tuberin phosphorylation and OGG1 expression in primary cultures of RPTE cells. A: Representative immunoblot shows an increase in phospho-Akt (p-Akt) and phospho-tuberin (p-tuberin) and a decrease in OGG1 expression in cells treated with high glucose for the time periods indicated. GAPDH was used as loading control. B: DCF fluorescence was measured using the peroxide-sensitive fluorescent probe DCF-DA by a multiwell fluorescence plate reader in intact RPTE cells treated with high glucose. Histogram represents means ± SE of three independent experiments. Significant difference from nontreated cells is indicated by *P < 0.05 and **P < 0.01.
FIG. 9.
FIG. 9.
Proposed model of oxidative DNA damage in diabetes.
See this image and copyright information in PMC

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