Epac is required for GLP-1R-mediated inhibition of oxidative stress and apoptosis in cardiomyocytes

Abstract

Although the cardioprotective effects of glucagon-like peptide-1 and its analogs have been reported, the exact mechanisms of the glucagon-like peptide-1 receptor (GLP-1R) signaling pathway in the heart are still unclear. Activation of the GLP-1R has been shown to increase cAMP levels, thus eliciting protein kinase A- and exchange protein activated by cAMP (Epac)-dependent signaling pathways in pancreatic β-cells. However, which pathway plays an important role in the antioxidant and antiapoptotic effects of GLP-1R activation in the heart is not known. In this study, we demonstrated that stimulation of GLP-1Rs with exendin-4 attenuated H2O2-induced reactive oxygen species production and increased the synthesis of antioxidant enzymes, catalase, glutathione peroxidase-1, and manganese superoxide dismutase that is dependent on Epac. Additionally, exendin-4 has an antiapoptotic effect by decreasing a number of apoptotic cells, inhibiting caspase-3 activity, and enhancing the expression of antiapoptotic protein B-cell lymphoma 2, which is mediated through both protein kinase A- and Epac-dependent pathways. These data indicate a critical role for Epac in GLP-1R-mediated cardioprotection.

Figure 1.. Exendin-4 inhibits H₂O₂-induced oxidative stress and apoptosis.

A and B, Cells were pretreated without or with 100 nM exendin-(9–39) (9–39) for 1 hour before treatment with vehicle (control), 100 μM vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 μM H₂O₂ for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. B, DCF fluorescence is shown in green color. Scale bar, 10 μm (n = 4). C and D, Cells were pretreated without or with 100 nM exendin-(9–39) for 1 hour before treatment with vehicle or 20 nM Ex-4 for 3 hours. C, Cells were then incubated with 250 nM H₂O₂ for 24 hours (C) or 200 μM H₂O₂ for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ± SEM. Scale bar, 10 μm (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂.

Figure 2.. Antioxidant and antiapoptotic effects of exendin-4 is cAMP dependent.

A and B, Cells were pretreated without or with 1 μM ddA (an AC inhibitor) for 1 hour before treatment with vehicle (control), 0.1 μM or 10 μM forskolin (Forsk; AC activator), 100 μM vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 μM H₂O₂ for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. B, DCF fluorescence is shown in green color. Scale bar, 10 μm (n = 4). C and D, Cells were pretreated without or with 1 μM ddA before treatment with vehicle (control), 0.1 μM or 10 μM forskolin (Forsk; an AC activator), or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H₂O₂ for 24 hours (C) or 200 μM H₂O₂ for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ± SEM. Scale bar, 10 μm (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 v. H₂O₂.

Figure 3.. Exendin-4 prevents H₂O₂-mediated apoptosis in a PKA-dependent pathway.

A and B, Cells were pretreated without or with 10 μM PKI (a PKA inhibitor) for 1 hour before treatment with vehicle (control), 1 μM 6-Benz-cAMP (a PKA activator), 100 μM vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 μM H₂O₂ for 30 minutes. A, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. B, DCF fluorescence is shown in green color. Scale bar, 10 μm (n = 4). C and D, Cells were pretreated without or with 10 μM PKI for 1 hour before treatment with vehicle, 1 μM 6-Benz-cAMP, 100 μM vitamin C, or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H₂O₂ for 24 hours (C) or 200 μM H₂O₂ for 3 hours (D). C, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ± SEM. Scale bar, 10 μm (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. D, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. E, Cell lysates were immunoblotted with antiphospho-CREB and anti-CREB antibodies. The CREB activation was quantified, expressed as fold increase over control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control.

Figure 4.. Exendin-4 prevents H₂O₂-mediated oxidative stress and apoptosis in an Epac-dependent pathway.

A, Cells were transfected with 100 nM Epac siRNA or control siRNA. The fold increase in mRNA expression of Epac gene are calculated from 2^−ΔΔCT. The mRNA levels were quantified and shown as the mean ± SEM (n = 4). *, P < .05 vs control siRNA. B and C, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were treated with vehicle (control), 10 μM ESCA-AM (Epac selective cAMP analog acetoxymethyl ester), 100 μM vitamin C, or 20 nM exendin-4 (Ex-4) for 3 hours. Cells were then incubated with 200 μM H₂O₂ for 30 minutes. B, The intracellular ROS production was quantified, expressed as a percentage of the control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂. C, DCF fluorescence is shown in green color. Scale bar, 10 μm (n = 4). D and E, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 10 μM PKI for 1 hour before treatment with vehicle, 5 μM ESCA-AM, or 20 nM Ex-4 for 3 hours. Cells were then incubated with 250 nM H₂O₂ for 24 hours (C) or 200 μM H₂O₂ for 3 hours (D). D, Apoptotic cells were assessed by TUNEL stain (green) and cells counterstained with DAPI (blue) to show cell nuclei. The values are expressed as the percentage of apoptotic cells over nonstimulated cells and shown as the mean ± SEM. Scale bar, 10 μm (n = 4). *, P < .05 vs control; #, P < .05 vs the H₂O₂. E, The caspase-3 activity was quantified, expressed as the value of absorbance, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs H₂O₂.

Figure 5.. Effects of exendin-4 on the mRNA and protein expressions of antioxidant enzymes.

A, Cells were stimulated with 20 nM exendin-4 (Ex-4) for 6 hours. The relative mRNA levels were quantified and shown as the mean ± SEM (n = 4). *, P < .05 vs vehicle. B, Cells were stimulated with 20 nM Ex-4 for 24 hours. Cell lysates were immunoblotted with anti-GPx-1, anti-Mn-SOD, anticatalase, and anti-GAPDH antibodies. The relative protein levels were quantified, expressed as fold increase over control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control.

Figure 6.. Stimulation of GLP-1R enhances the mRNA and protein expressions of antioxidant enzymes in an Epac-dependent manner.

A and B, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 1 μM ddA (an AC inhibitor), 10 μM PKI (a PKA inhibitor), or 5 μM KN-93 (a CaMKII inhibitor) for 1 hour before treatment with vehicle (control) or 20 nM exendin-4 (Ex-4) for 6 hours (A) or 24 hours (B). A, The relative mRNA levels were quantified and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs Ex-4-treated control siRNA. B, The relative protein levels were quantified, expressed as fold increase over control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control.

Figure 7.. Stimulation of GLP-1R induces the mRNA and protein expressions of antiapoptotic protein in PKA- and Epac-dependent manners.

A and B, Cells were stimulated with 20 nM exendin-4 (Ex-4) for 6 hours. The relative mRNA levels were quantified and shown as the mean ± SEM (n = 4). *, P < .05 vs vehicle. C, Cells were stimulated with 20 nM Ex-4 for 24 hours. Cell lysates were immunoblotted with anti-Bcl-2 and anti-GAPDH antibodies. The relative protein levels were quantified, expressed as fold increase over control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control. D and E, Cells were transfected with either Epac siRNA or control siRNA. After 2 days, cells were pretreated without or with 1 μM ddA (an AC inhibitor), 10 μM PKI (a PKA inhibitor) or 5 μM KN-93 (a CaMKII inhibitor) for 1 hour before treatment with vehicle (control) or 20 nM exendin-4 (Ex-4) for 6 hours (D) or 24 hours (E). D, The relative Bcl-2 mRNA level was quantified and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs Ex-4-treated control siRNA. E, The relative Bcl-2 protein level was quantified, expressed as fold increase over control, and shown as the mean ± SEM (n = 4). *, P < .05 vs control; #, P < .05 vs Ex-4-treated control siRNA.

Figure 8.. Schematic diagram representing the cardioprotective effects of exendin-4.

Exendin-4 (Ex-4) binding to GLP-1Rs leads to G_s protein coupling, subsequently stimulates AC activity, and generates cAMP, the second messenger. cAMP can bind to and activate both PKA and Epac signaling. Stimulation of GLP-1Rs results in the antioxidation by inducing the synthesis of catalase, GPx-1, and Mn-SOD through Epac signaling and elicits the antiapoptosis by enhancing Bcl-2 production and inhibiting caspase-3 activity via PKA and Epac signalings.