Introduction: We had found GLP-1(28-36), a neutral endopeptidase-derived metabolite of glucagon-like peptide-1 (GLP-1), to be as protective against ischemia-reperfusion injury (IRI) as GLP-1 in ex vivo isolated heart and in vivo myocardial infarction (MI) models in mice, and that this effect required soluble adenylyl cyclase (sAC). We had also found that GLP-1(28-36) augments ATP-dependent sAC activity and cAMP production in coronary vascular cells but not cardiomyocytes. Here we elucidate the mechanism by which GLP-1(28-36) activates sAC.
Methods & Results: Affinity pull-down using biotinylated-GLP-1(28-36), proteomics, and confirmatory immunoblots showed that GLP-1(28-36) interacts with mitochondrial trifunctional protein-α (MTPα), an enzyme involved in fatty acid β-oxidation, and does not interact with sAC. We next used Seahorse XFe24 extracellular flux analysis for glycolysis- and mitochondrial-stress tests, and energy substrate (glucose, palmitic acid) oxidation assays in coronary artery smooth muscle cells (caSMC) and human coronary artery endothelial cells (caEC). Treatment with GLP-1(28-36) increased glycolysis by 36% and 43%, glycolytic capacity by 35% and 54% in caSMC and caEC respectively, and mitochondrial glucose oxidation by 26% in caEC, as compared to treatment with scrambled peptide control [Scram(28-36)] (P<0.05 for all comparisons). Importantly, compared to Scram(28-36), caSMC treated with GLP-1(28-36) revealed a 72% decrease in palmitic acid oxidation (P<0.001). To test if these effects depend on MTPα, we repeated these experiments after siRNA-mediated knockdown of MTPα mRNA (76%) and protein (67%). At baseline, cells transfected with MTPα siRNA showed a 20% reduction in palmitic acid oxidation, and 30% and 33% increases in glycolysis and glucose oxidation, respectively compared to control siRNA (P<0.05 for all comparisons). Importantly, the ability of GLP-1(28-36) to reduce palmitic acid oxidation was lost in MTPα siRNA-treated cells, but not in control siRNA-treated cells. Together, these results indicate that GLP-1(28-36) shifts substrate preference from fatty acid to glucose utilization by inhibiting MTPα.
Conclusion: GLP-1(28-36) prevents cardiac IRI by inhibiting MTPα in coronary vascular cells.