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).