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

Simone S, Gorin Y, Velagapudi C, Abboud HE, Habib SL - Diabetes (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: George O'Brien Kidney Research Center, Department of Medicine, Division of Nephrology, University of Texas Health Science Center, San Antonio, Texas, USA.

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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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.)
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f7: 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.)

Mentions: High glucose increases ROS production in vascular cells and in renal cells, including tubular epithelial cells or mesangial cells (2,4,41). The fact that oxidant-induced effects on Akt and tuberin phosphorylation correlate well with those of high glucose suggests that ROS may mediate the action of high glucose on Akt, tuberin, and OGG1. First, we evaluated the role of high glucose in the generation of intracellular ROS. MCT cells were incubated for the indicated time periods in serum-free medium containing either normal glucose or high glucose. High glucose significantly increased the fluorescence of DCF, a peroxide-sensitive probe, with a maximal effect apparent 60 min after treatment, as measured by confocal microscopy (Fig. 7A). Additionally, measurements were performed with a multiwell fluorescence plate reader to provide a better quantification of high glucose–induced ROS generation. Incubation of MCT cells with high glucose resulted in a rapid and time-dependent increase in DCF fluorescence seen as early as 5 min after treatment (Fig. 7B). These findings demonstrate that high glucose increases intracellular ROS generation in MCT cells. Next, we assessed whether ROS generation mediates the effect of high glucose–induced Akt and tuberin phosphorylation and OGG1 downregulation in MCT cells. Serum-starved cells were pretreated with the antioxidant NAC, a ROS scavenger. Pretreatment with 20 mmol/l NAC significantly inhibited ROS generation measured by DCF fluorescence in cells exposed to high glucose for 30 min (Fig. 7C). Moreover, pretreatment of the cells with NAC inhibited Akt and tuberin phosphorylation and upregulation of OGG1 protein expression in cells exposed to high glucose (Fig. 7D). Furthermore, pretreatment of the cells with NAC significantly reduced 8-oxodG staining compared with cells treated with high glucose alone (Fig. 7E). These data indicate that ROS are critical for high glucose–induced Akt and tuberin phosphorylation and downregulation of OGG1. High glucose increases Akt and tuberin phosphorylation and downregulates OGG1 in primary proximal tubular epithelial cells. To confirm the validity of these data obtained in the MCT immortalized cells, we studied the effects of high glucose on the phosphorylation of Akt and tuberin and on OGG1 protein expression in RPTE cells. Serum-deprived RPTE cells were treated with 25 mmol/l glucose for different periods of time. High glucose concentration caused an increase in phosphorylation of Akt and tuberin, with a peak at 30 min and subsiding by 60 min, compared with cells incubated with 5 mmol/l glucose (Fig. 8A). Moreover, the increase in phosphorylated Akt and phosphorylated tuberin is associated with a decrease in OGG1 expression in the cells incubated with 25 mmol/l glucose for 60 min (Fig. 8A). These data confirm our observation in the MCT immortalized cells.


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

Simone S, Gorin Y, Velagapudi C, Abboud HE, Habib SL - Diabetes (2008)

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.)
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f7: 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.)
Mentions: High glucose increases ROS production in vascular cells and in renal cells, including tubular epithelial cells or mesangial cells (2,4,41). The fact that oxidant-induced effects on Akt and tuberin phosphorylation correlate well with those of high glucose suggests that ROS may mediate the action of high glucose on Akt, tuberin, and OGG1. First, we evaluated the role of high glucose in the generation of intracellular ROS. MCT cells were incubated for the indicated time periods in serum-free medium containing either normal glucose or high glucose. High glucose significantly increased the fluorescence of DCF, a peroxide-sensitive probe, with a maximal effect apparent 60 min after treatment, as measured by confocal microscopy (Fig. 7A). Additionally, measurements were performed with a multiwell fluorescence plate reader to provide a better quantification of high glucose–induced ROS generation. Incubation of MCT cells with high glucose resulted in a rapid and time-dependent increase in DCF fluorescence seen as early as 5 min after treatment (Fig. 7B). These findings demonstrate that high glucose increases intracellular ROS generation in MCT cells. Next, we assessed whether ROS generation mediates the effect of high glucose–induced Akt and tuberin phosphorylation and OGG1 downregulation in MCT cells. Serum-starved cells were pretreated with the antioxidant NAC, a ROS scavenger. Pretreatment with 20 mmol/l NAC significantly inhibited ROS generation measured by DCF fluorescence in cells exposed to high glucose for 30 min (Fig. 7C). Moreover, pretreatment of the cells with NAC inhibited Akt and tuberin phosphorylation and upregulation of OGG1 protein expression in cells exposed to high glucose (Fig. 7D). Furthermore, pretreatment of the cells with NAC significantly reduced 8-oxodG staining compared with cells treated with high glucose alone (Fig. 7E). These data indicate that ROS are critical for high glucose–induced Akt and tuberin phosphorylation and downregulation of OGG1. High glucose increases Akt and tuberin phosphorylation and downregulates OGG1 in primary proximal tubular epithelial cells. To confirm the validity of these data obtained in the MCT immortalized cells, we studied the effects of high glucose on the phosphorylation of Akt and tuberin and on OGG1 protein expression in RPTE cells. Serum-deprived RPTE cells were treated with 25 mmol/l glucose for different periods of time. High glucose concentration caused an increase in phosphorylation of Akt and tuberin, with a peak at 30 min and subsiding by 60 min, compared with cells incubated with 5 mmol/l glucose (Fig. 8A). Moreover, the increase in phosphorylated Akt and phosphorylated tuberin is associated with a decrease in OGG1 expression in the cells incubated with 25 mmol/l glucose for 60 min (Fig. 8A). These data confirm our observation in the MCT immortalized cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: George O'Brien Kidney Research Center, Department of Medicine, Division of Nephrology, University of Texas Health Science Center, San Antonio, Texas, USA.

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.

Show MeSH
Related in: MedlinePlus