Hyperglycemia-induced activation of the hexosamine biosynthetic pathway causes myocardial cell death

[摘要] ENGLISH ABSTRACT: OBJECTIVE – Oxidative stress increases flux through the hexosamine biosynthetic pathway(HBP) resulting in greater O-GlcNAcylation of target proteins. Since increased oxidative stressand HBP flux are associated with insulin resistance, we hypothesized that its activation leads togreater O-GlcNAcylation of BAD (pro-apoptotic) and increased myocardial apoptosis.RESEARCH DESIGN AND METHODS – To investigate our hypothesis, we employed twoexperimental models: 1) H9c2 cardiomyoblasts exposed to high glucose (33 mM glucose) ± HBPmodulators ± antioxidant treatment vs. matched controls (5.5 mM glucose); and 2) a rat model ofhigh fat diet-induced insulin resistance and hyperglycemia. We evaluated apoptosis in vitro byHoechst nuclear staining, Annexin-V staining, caspase activity measurements andimmunoblotting while in vivo apoptosis was assessed by immunoblotting. In vitro reactiveoxygen species (ROS) levels were quantified by H2DCFDA staining (fluorescence microscopy,flow cytometry). We determined overall and BAD O-GlcNAcylation, both by immunoblottingand immunofluorescence microscopy. As BAD-Bcl-2 dimer formation enhances apoptosis, weperformed immunoprecipitation analysis and immunofluorescence microscopy (co-localization)to determine BAD-cl-2 dimerization. In vivo overall O-GlcNAcylation, BAD O-GlcNAcylationand BAD-Bcl-2 dimerization was determined by immunoprecipitation and immunoblotting. 4RESULTS – High glucose treatment of cells significantly increased the degree of apoptosis asrevealed by Hoechst nuclear staining (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V staining (43 ±5%), caspase activity assay (26 ± 2%) and immunoblotting. In parallel, overall OGlcNAcylation(p<0.001 vs. 5.5 mM), BAD O-GlcNAcylation (p<0.05 vs. 5.5 mM) and ROSlevels were increased (fluorescence microscopy – p<0.05 vs. 5.5 mM; flow cytometry – p<0.001vs. 5.5 mM). HBP inhibition using DON and antioxidant treatment (α-OHCA) attenuated theseeffects while HBP activation by PUGNAc exacerbated it. Likewise, insulin resistant rat heartsexhibited significantly higher caspase-3 (p<0.05 vs. controls), overall O-GlcNAcylation (p<0.05vs. controls) and BAD O-GlcNAcylation levels (p<0.05 vs. 5.5 mM). BAD-Bcl-2 dimerformation was increased in cells exposed to hyperglycemia [immunoprecipitation analysis andco-localization] and in insulin resistant hearts.CONCLUSIONS - Our study identified a novel pathway whereby hyperglycemia results ingreater oxidative stress, resulting in increased HBP activation and increased BAD OGlcNAcylation.We also found greater BAD-Bcl-2 dimerization increasing myocardialapoptosis, suggesting that this pathway may play a crucial role in the onset of the diabeticcardiomyopathy.

[发布机构] Stellenbosch University