The hexosamine biosynthetic pathway can mediate myocardial apoptosis in a rat model of diet-induced insulin resistance

Aims: Type 2 diabetes is characterized by deranged metabolic pathways that may result in cardiovascular complications. For example, hyperglycaemia promotes flux through the hexosamine biosynthetic pathway (HBP) leading to greater O-GlcNAcylation of target proteins, with pathophysiological outcomes. This study investigated mechanisms whereby increased HBP flux elicits myocardial apoptosis in a rat model of diet-induced hyperglycaemia/insulin resistance. Methods: Four-week-old male Wistar rats were fed a high-fat diet (86 days) after which insulin resistance was assessed vs. matched controls. Oxidative stress was evaluated, and apoptotic peptide levels, BAD phosphorylation and overall O-GlcNAcylation assessed by immunoblotting. Protein-specific O-GlcNAcylation and BAD-Bcl-2 dimerization were determined by immunoprecipitation and Western blotting. Results: Rats consuming the high-fat diet exhibited a moderate elevation in body weight, higher fasting insulin and glucose levels, and insulin resistance vs. controls. Overall protein O-GlcNAcylation was increased in hyperglycaemic/insulin-resistant hearts. In parallel, myocardial peptide levels of apoptotic markers (caspase-3, cytochrome-c, BAD) were significantly higher with insulin resistance. To gain mechanistic insight into our findings, we evaluated O-GlcNAcylation of BAD, a pro-apoptotic Bcl-2 homolog. Here we found increased BAD O-GlcNAcylation and decreased BAD phosphorylation (Ser136) in hyperglycaemic/insulin-resistant rat hearts. These data are in agreement with competition by phosphorylation and O-GlcNAcylation for the same or neighbouring site(s) on target proteins. Moreover, we observed increased BAD-Bcl-2 dimerization in hyperglycaemic/insulin-resistant hearts. Conclusion: The main finding of this study is that increased apoptosis in hyperglycaemic/insulin-resistant hearts can also be mediated through HBP-induced BAD O-GlcNAcylation and greater formation of BAD-Bcl-2 dimers (pro-apoptotic).