Cardioprotective effects of GLP-1 7-36 amide and its major circulating metabolite, GLP-1 9-36 amide.

N Storey, S Afreen, F Mutongwizo, G Willars. University of Leicester, Leicestershire, UK

Cardioprotection is a phenomenon whereby damage to the myocardium caused by ischaemia and subsequent reperfusion can be ameliorated. Such protection can be conferred by ischaemic preconditioning, mitochondrial uncouplers (eg. dinitrophenol; DNP), and hormones, including glucagon-like-peptide-1 (GLP-1) (Timmers et al., 2009). GLP-1 7-36 amide is the major circulating postprandial form of GLP-1 and enhances glucose-dependent insulin release through the Gαs coupled GLP-1 receptor (GLP-1R) making the receptor an attractive target for the treatment of type 2 diabetes. The metabolism of GLP-1 (t1/2 ~2 min) rapidly produces metabolites, particularly GLP-1 9-36 amide, which is thought to terminate the biological effects of GLP-1 although it has also been reported as a low potency partial agonist. Here we have investigated the cardioprotective effects of GLP-1 7-36 amide and GLP-1 9-36 amide and explored cardioprotective signalling pathways.

Adult Wistar rat (~300g) cardiac myocytes were isolated using the Langendorff method (Rodrigo et al., 2002). Ischaemia/reperfusion was simulated by 7-min superfusion with metabolic inhibition (MI) solution (2mM cyanide and 1mM iodoacetic acid to inhibit ATP synthesis) and 10-min re-energisation with Tyrode’s to induce reperfusion injury. Myocytes were electrically-field stimulated (1Hz) and contractile myocytes counted after 10-minutes re-energisation. Data are %±SEM from n myocytes, n experiments and n hearts with statistical analysis by ANOVA and Dunnett’s test.

GLP-1R in myocytes was confirmed by PCR. Following MI, 35±2% of control myocytes recovered contractile function (571, 16, 8). Pretreatment (5-min) with DNP (5µM), GLP-1 7-36 amide or GLP-1 9-36 amide (300nM) significantly (P<0.05) increased contractile recovery (DNP 64±4% (302, 10, 8); GLP-1 7-36 amide 58±2% (194, 6, 4) and; GLP-1 9-36 amide 60±2% (252, 6, 6)). Application of either dibutyryl-cAMP (500µM) or the EPAC (exchange protein directly activated by cAMP) activator 8-pCPT-2′-O-Me-cAMP (8-(4-chlorophenylthio)-2\'-O-methyladenosine-3\',5\'-cyclic monophosphate acetoxymethyl ester) (50µM) also significantly (P<0.001) increased contractile recovery (dibutyryl--cAMP 62±8% (117, 4, 3 hearts); 8-p-CPT 61±8% (179, 5, 3)). The protein kinase A inhibitors H89 (1µM) and PKi 14-22 amide, myristoylated (1µM) blocked (P<0.001) the cardioprotective effect of GLP-1 9-36 amide but not that of GLP-1 7-36 amide.

In summary, GLP-1 7-36 amide and GLP-1 9-36 amide exerted cardioprotetive effects on isolated mycoytes in a model of ischaemia reperfusion injury. The cAMP analogue dibutyryl-cAMP and the EPAC activator 8-pCPT-2′-O-Me-cAMP mimicked the cardioprotective effects of GLP-1. The effects of GLP-1 9-36 amide but not GLP-1 7-36 amide were protein kinase A-dependent.

Timmers LJ et al, Am Coll Cardiol 53:501, 2009

Rodrigo GC et al, J Mol Cell Cardiol 34:555, 2002