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Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial apoptosis

Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial... Abstract The mechanisms mediating hyperglycemia-mediated myocardial cell death are poorly defined. Since elevated flux through the hexosamine biosynthetic pathway (HBP) is closely linked with the diabetic phenotype, we hypothesized that hyperglycemia-mediated oxidative stress results in greater O -GlcNAcylation (HBP end product) of the proapoptotic peptide BAD, thereby increasing myocardial apoptosis. H9c2 cardiomyoblasts were exposed to high glucose (33 mM) ± HBP modulators ± antioxidant treatment for 5 days vs. matched controls (5.5 mM), and we subsequently evaluated apoptosis by immunoblotting, immunofluorescence staining, and caspase activity measurements. In vitro reactive oxygen species (ROS) levels were quantified by 2′,7′-dichlorodihydrofluorescein diacetate staining (fluorescence microscopy and flow cytometry). We determined total and BAD O -GlcNAcylation, respectively, by immunoblotting and immunofluorescence microscopy. The current study shows that high glucose treatment of cells significantly increased the degree of apoptosis. In parallel, overall O -GlcNAcylation, BAD O -GlcNAcylation, and ROS levels were increased. HBP inhibition and antioxidant treatment attenuated these effects, while increased end product levels exacerbated it. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and colocalization and found increased dimerization in cells exposed to hyperglycemia. Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress and increased HBP activation and BAD O -GlcNAcylation in H9c2 cardiomyoblasts. Since greater BAD-Bcl-2 dimerization increases myocardial apoptosis, this pathway may play a crucial role in diabetes-related onset of heart diseases. oxidative stress heart cell death O -GlcNAc Copyright © 2010 the American Physiological Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AJP - Cell Physiology The American Physiological Society

Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial apoptosis

Rajamani, Uthra; Essop, M. Faadiel
AJP - Cell Physiology , Volume 299 (1): C139 – Jul 1, 2010
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Publisher
The American Physiological Society
Copyright
Copyright © 2010 the American Physiological Society
ISSN
0363-6143
eISSN
1522-1563
DOI
10.1152/ajpcell.00020.2010
pmid
20410435
Publisher site
See Article on Publisher Site

Abstract

Abstract The mechanisms mediating hyperglycemia-mediated myocardial cell death are poorly defined. Since elevated flux through the hexosamine biosynthetic pathway (HBP) is closely linked with the diabetic phenotype, we hypothesized that hyperglycemia-mediated oxidative stress results in greater O -GlcNAcylation (HBP end product) of the proapoptotic peptide BAD, thereby increasing myocardial apoptosis. H9c2 cardiomyoblasts were exposed to high glucose (33 mM) ± HBP modulators ± antioxidant treatment for 5 days vs. matched controls (5.5 mM), and we subsequently evaluated apoptosis by immunoblotting, immunofluorescence staining, and caspase activity measurements. In vitro reactive oxygen species (ROS) levels were quantified by 2′,7′-dichlorodihydrofluorescein diacetate staining (fluorescence microscopy and flow cytometry). We determined total and BAD O -GlcNAcylation, respectively, by immunoblotting and immunofluorescence microscopy. The current study shows that high glucose treatment of cells significantly increased the degree of apoptosis. In parallel, overall O -GlcNAcylation, BAD O -GlcNAcylation, and ROS levels were increased. HBP inhibition and antioxidant treatment attenuated these effects, while increased end product levels exacerbated it. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and colocalization and found increased dimerization in cells exposed to hyperglycemia. Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress and increased HBP activation and BAD O -GlcNAcylation in H9c2 cardiomyoblasts. Since greater BAD-Bcl-2 dimerization increases myocardial apoptosis, this pathway may play a crucial role in diabetes-related onset of heart diseases. oxidative stress heart cell death O -GlcNAc Copyright © 2010 the American Physiological Society

Journal

AJP - Cell Physiology – The American Physiological Society

Published: Jul 1, 2010

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