title: Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial apoptosis.
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.
authors.affiliation: Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa.
authors.forename: Uthra
authors.initials: U
authors.lastname: Rajamani
authors.forename: M Faadiel
authors.initials: MF
authors.lastname: Essop
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053898:Biosynthetic Pathways; D002460:Cell Line; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D006595:Hexosamines / Q000096:biosynthesis*; D006943:Hyperglycemia / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019884:Nitrogenous Group Transferases / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D015854:Up-Regulation; D051025:bcl-Associated Death Protein / Q000378:metabolism
chemical_list: D000975:Antioxidants; C494821:Bad protein, rat; D006595:Hexosamines; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051025:bcl-Associated Death Protein; D019884:Nitrogenous Group Transferases; D005947:Glucose