Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α
M. Ahsan Siraj, … , Peter Backx, Mansoor Husain
M. Ahsan Siraj, … , Peter Backx, Mansoor Husain
Published January 27, 2020
Citation Information: J Clin Invest. 2020;130(3):1392-1404. https://doi.org/10.1172/JCI99934.
View: Text | PDF
Research Article Cardiology Metabolism

Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α

Abstract

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28–36), a neutral endopeptidase–generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28–36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10–dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28–36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC–/– cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28–36) and demonstrate that the ability of GLP-1(28–36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28–36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

Authors

M. Ahsan Siraj, Dhanwantee Mundil, Sanja Beca, Abdul Momen, Eric A. Shikatani, Talat Afroze, Xuetao Sun, Ying Liu, Siavash Ghaffari, Warren Lee, Michael B. Wheeler, Gordon Keller, Peter Backx, Mansoor Husain

×

Figure 3

GLP-1(28–36) stimulates intracellular cAMP accumulation, activates PKA, and prevents cytotoxicity in vascular cells.

Options: View larger image (or click on image) Download as PowerPoint
GLP-1(28–36) stimulates intracellular cAMP accumulation, activates PKA, ...
(A and B) Intracellular cAMP accumulation measured by enzymatic immunoassay in lysates from (A) caSMCs and (B) mouse and human caECs treated for 10 minutes with 100 nM each of GLP-1(28–36), scrambled control, IPE, forskolin, or PBS control (n = 3/treatment, each in triplicate). (C) Mouse caSMCs were pretreated with 100 nM GLP-1(28–36) or scrambled control for 20 minutes followed by incubation with H2O2 (100 μM) for 48 hours to induce oxidative stress (n = 3/each, in triplicate). LDH release was assayed by ELISA in duplicates from aliquots of cell culture media. (D) Mouse caSMCs were treated as above and lysed with PKA lysis buffer, with PKA activity determined by separation of phosphorylated (+ p-Peptag) and nonphosphorylated (– p-Peptag) substrate using electrophoresis. Densitometric analysis of phosphorylated bands showed increased PKA activity with GLP-1(28–36) as compared with the scrambled control. The blot is representative of 3 independent experiments. Max, maximum. (E) Whole-cell lysates were extracted from mouse caSMCs undergoing a similar 20-minute peptide pretreatment schedule, and apoptosis was assessed by cleaved caspase-3 activation at the end of the 7-hour incubation with or without H2O2 (100 μM). Representative Western blot and normalized densitometric analysis (n = 3/group) show that GLP-1(28–36) prevented activation of cleaved caspase-3 in the presence of H2O2 versus controls. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA (except for data in E, which were analyzed by 2-way ANOVA); NS versus the corresponding control with Bonferroni’s post hoc test.