Glucagon-like peptide-1 protects cardiomyocytes from advanced oxidation protein product-induced apoptosis via the PI3K/Akt/Bad signaling pathway

Abstract

Cardiomyocyte apoptosis is a major event in the pathogenesis of diabetic cardiomyopathy. Currently, no single effective treatment for diabetic cardiomyopathy exists. The present study investigated whether advanced oxidative protein products (AOPPs) have a detrimental role in the survival of cardiomyocytes and if glucagon-like peptide-1 (GLP-1) exerts a cardioprotective effect under these circumstances. The present study also aimed to determine the underlying mechanisms. H9c2 cells were exposed to increasing concentrations of AOPPs in the presence or absence of GLP-1, and the viability and apoptotic rate were detected using a cell counting kit-8 assay and flow cytometry, respectively. In addition, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor, LY294002, was employed to illustrate the mechanism of the antiapoptotic effect of GLP-1. The expression levels of the apoptotic-associated proteins, Akt, B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter (Bad), Bcl-2-associated X protein (Bax) and caspase-3 were measured by western blotting. It was revealed that GLP-1 significantly attenuated AOPP-induced cell toxicity and apoptosis. AOPPs inactivated the phosphorylation of Akt, reduced the phosphorylation of Bad, decreased the expression of Bcl-2, increased the expression of Bax and the activation of caspase-3 in H9c2 cells. GLP-1 reversed the above changes induced by AOPPs and the protective effects of GLP-1 were abolished by the PI3K inhibitor, LY294002. In conclusion, the present data suggested that GLP-1 protected cardiomyocytes against AOPP-induced apoptosis, predominantly via the PI3K/Akt/Bad pathway. These results provided a conceivable mechanism for the development of diabetic cardiomyopathy and rendered a novel application of GLP-1 exerting favorable cardiac effects for the treatment of diabetic cardiomyopathy.

Figure 1

GLP-1 attenuated AOPPs-RSA-induced toxicity and apoptosis in H9c2 cells. The cell viability, morphological changes and apoptotic rates were assessed using a cell counting kit-8 assay, Hoechst 33258 staining and flow cytometry, respectively. (A) The cells were incubated with RSA (1 µg/ml), or increasing concentrations of AOPPs-RSA for 24 h. AOPPs-RSA inhibited cell viability in a dose-dependent manner. (B) The cells were incubated with 1 µg/ml AOPPs-RSA for 24 h in the presence or absence of different concentrations of GLP-1. GLP-1 improved cell viability in a dose-dependent manner. (C) The morphological changes in H9c2 cells were detected using a fluorescence microscope. Chromatin condensation and nuclear fragmentation represented apoptotic cells, while the diffuse blue fluorescence represented normal cells (magnification, ×200). (D) The apoptotic rate of each treatment was determined. The data are expressed as the mean ± standard deviation of three independent experiments (^*P<0.05 and ^**P<0.01, vs. control; ^#P<0.05 and ^##P<0.01, vs. AOPPs-treated cells; ^&&P<0.01, vs. AOPPs + GLP-1-treated group). GLP-1, Glucagon-like peptide-1; AOPPs, advanced oxidation protein products; RSA, rat serum albumin.

Figure 2

GLP-1 downregulates the expression of RAGE in H9c2 cells. (A) H9c2 cells were immunostained with anti-mouse RAGE primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green), and were counterstained with DAPI (blue). The expression of RAGE was upregulated by exposure of increasing concentrations of AOPPs-RSA for 24 h (magnification, ×400). (B) GLP-1 significantly decreased the protein expression of RAGE, as determined by western blotting. (C) GLP-1 significantly decreased the mRNA expression of RAGE as determined by reverse transcription-quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation of three independent experiments (^**P<0.01, vs. control; ^##P<0.01, vs. AOPPs-RSA-treated cells). GLP-1, Glucagon-like peptide-1; AOPPs, advanced oxidation protein products; RSA, rat serum albumin; RAGE, receptor for advanced glycation end product; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

GLP-1 exerts its protective function via the GLP-1 receptor in H9c2 cells. (A) H9c2 cells were immunostained with anti-rabbit GLP-1R primary antibody and fluorescein isothiocyanate-conjugated secondary antibody (green), and counterstained with DAPI (blue; magnification, ×200). (B) GLP-1 significantly increased the expression of the GLP-1R, as determined by western blot analysis. (C) The mRNA expression of GLP-1R was increased following exposure to GLP-1 (50 nM) for 24 h, as measured by reverse transcription-quantitative polymerase chain reaction. The data are expressed as the mean ± standard deviation of three independent experiments (^**P<0.01, vs. control; ^##P<0.01, vs. AOPPs-treated cells). GLP-1, Glucagon-like peptide-1; GLP-1R, GLP-1 receptor; AOPPs, advanced oxidation protein products; RSA, rat serum albumin; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4

GLP-1 prevents AOPPs-induced apoptosis via the PI3K/Akt/BAD pathway in H9c2 cells. The cells were exposed to AOPPs-RSA (1 µg/ml), GLP-1 (50 nM) or LY294002 (10 µM) for 24 h. The protein expression levels were detected by western blotting. GLP-1 reversed AOPPs-RSA-induced (A) p-Akt and (B) p-bad inactivation. LY294002 significantly inhibited the effect of GLP-1. AOPPs-RSA decreased the expression of (C) Bcl-2 and increased the expression of (D) Bax and (E) activated caspase-3. GLP-1 reversed these effects, however, was suppressed by LY294002. The data are expressed as the mean ± standard deviation of three independent experiments (^*P<0.05 and ^**P<0.01, vs. control; ^#P<0.05 and ^##P<0.01, vs. AOPPSs-RSA treated cells). GLP-1, Glucagon-like peptide-1; AOPPs, advanced oxidation protein products; RSA, rat serum albumin; p-, phosphorylated; Bcl, B-cell lymphoma; Bax, Bcl-2-associated X protein; Bad, Bcl-2-associated death promoter.