In the present study, we provided for the first time that a truncated metabolite of GLP-1 hormone, GLP-1(9–36) rescued oxidative stress and apoptosis in H9c2 cardiomyoblasts through PI3K/Akt/NOS signaling pathway. This protective role is likely by ways of increasing the production of antioxidant enzymes, GPx-1, catalase, HO-1, inactivation of caspase-3 apoptotic pathway, and upregulation of antiapoptotic proteins, Bcl-2, Bcl-xL.
Since oxidative stress is primarily caused by imbalance between oxidants and antioxidants, overproduction of ROS can potentially trigger the processes of DNA fragmentation, protein and lipid oxidations and is considered as one of the major contributions to apoptosis and cell death27,28 (Kim and Kang, 2010). In the heart, oxidative stress and apoptosis are related to the pathological processes of ischemia and play an essential role in the progression and development of heart diseases28–30. H2O2 can act as the destructive molecule that involved in most of the redox reactions in the cells, and a high level of H2O2 is one of the major causes of myocardial I/R injury23. According to H2O2 is widely used in models for myocardial I/R injury which leads to reduced antioxidant activity and also induced cell death24,31, in the present study, we used H2O2 as a inducer of oxidative stress and apoptosis in H9c2 cells. Therefore, the inhibition of oxidative stress and apoptosis induced by H2O2 in cardiac cells may be an effective way to prevent myocardial I/R injury and improve cardiac function after cardiac injury.
GLP-1 and its analogs (GLP-1R agonists; exenatide, liraglutide) were intensively studied to find their cardioprotective effects against cardiac injury. GLP-1 is an incretin hormone which is produced in the gut in response to food intake. GLP-1 has two active isoform; GLP-1(7–36) and GLP-1(7–37)8,9. In the circulatory system, approximately 80% of total GLP-1 hormone is GLP-1(7–36), which is rapidly cleaved by enzyme DPP-4 to GLP-1(9–36)32. Due to GLP-1(9–36) has no significant effects on insulin secretion and weak interaction to GLP-1R, it was previously recognized as a truncated inactive metabolite of GLP-1(9–37)13. Over the past decade, accumulated reports have demonstrated the protective effects of GLP-1(9–36) in many tissues, including the heart. Treatment with both exendin-4 (GLP-1R agonist) and GLP-1(9–36) provoked protective effects against MG-induced mitochondrial dysfunction through GLP-1R-dependent and GLP-1R-independent manner, respectively in H9c2 cells12. In dog with dilated cardiomyopathy model, administration of GLP-1(9–36) induced myocardial glucose uptake and improved LV function16. In contrast to GLP-1(7–36), GLP-1(9–36) suppressed high glucose-induced superoxide production in human arterial endothelial cells15. In addition, GLP-1(9–36) ameliorated cell survival in response to I/R injury and hydrogen peroxide treatment in human aortic endothelial cells18. Moreover, GLP-1(9–36) suppressed mitochondrial ROS production induced by high glucose in human endothelial cells33. Consistently with these previous studies, we also observed that GLP-1(9–36) treatment strongly inhibited H2O2-induced ROS production and caspase-3 activity in H9c2 cardiomyoblasts. These data imply that GLP-1(9–36) possesses cardioprotective effects through inhibition of oxidative stress and apoptosis. Exendin-4 is a peptide isolated from the saliva of the gila monster lizard which shows about 53% amino acid sequence homology with GLP-1 hormone.
Exendin-4 is resistant to DPP-4 cleavage and acts as a potent GLP-1R agonist34. Interestingly, exendin-4 represented antioxidant and antipoptotic effects appeared to be greater than that of GLP-1(9–36) in H9c2 cardiomyoblasts against the same H2O2 concentration (Fig. 1). Indeed, the molecular mechanism for these effects of GLP-1(9–36) is different from that of GLP-1R agonist. GLP-1(9–36) is further cleaved by neutral endopeptidase to generate GLP-1(28–36)35. GLP-1(28–36) prevented myocardial ischemic injury and also reduced myocardial infarct size in mice model of ischemic injury36. However, it remains an unsolved question whether GLP-1(28–36), a cleavage product of GLP-1(9–36), exhibits antioxidant and antiapoptotic effects.
The death of cardiac cells from apoptosis causes a significant loss in cardiac functions. Therefore, several studies have focused on the ways to protect cardiac myocytes from pathological stimuli, including oxidative stress. Due to its importance in the cardiac contractility, cardiac myocyte seems to have defense mechanisms itself against oxidative stress37. One of these mechanisms of cardiac myocytes could be an induction of antioxidant enzyme production, including its activity. Various types of antioxidant enzymes (e.g., catalase, GRe, GPx, SOD, HO-1) are the major components for scavenging the reactive free radicals in many cell types. These antioxidant abilities of GLP-1 are believed to arise from enhancement of GLP-1 signaling leading to upregulation of antioxidant enzyme synthesis. For example, GLP-1 prevented ROS-mediated endothelial cell senescence by inducing the synthesis of HO-1 and NQO1 in HUVECs38. Stimulation of GLP-1R significantly inhibited oxidative stress in the liver of diabetic mice by upregulating catalase, GPx, and SOD39. These antioxidant mechanisms of GLP-1 are mediated through GLP-1 receptor-dependent manner. In the same way, antioxidant effects of GLP-1 cleavage product, GLP-1(9–36) might be due to the upregulation of antioxidant enzyme synthesis in cardiac cells. We found that GLP-1(9–36) leads to the significant upregulations of GPx-1, catalase, and HO-1 (Fig. 5). Interestingly, GLP-1(9–36)-mediated the upregulation of GPx-1, catalase, and HO-1 synthesis was not inhibited by either AC inhibitor or Epac inhibitor, indicating that the upregulation of these antioxidant enzymes by GLP-1(9–36) is independent of GLP-1R/cAMP/PKA signaling pathway in H9c2 cardiomyoblasts.
Apoptosis or death of cardiac cells is a key regulator in the pathogenesis of ischemic heart diseases especially after myocardial infarction, and the Bcl-2 family proteins are known as key regulators of apoptotic response. Bcl-2 family belongs to a group of apoptosis-regulating proteins and consists of two subgroups which are important to either inhibiting apoptosis (antiapoptotic proteins; Bcl-2, Bcl-xL, and Bcl-W) and promotes apoptosis (pro-apoptotic proteins; Bad, Bak, Bax, and Bid)40. Bcl-2 and Bcl-xL, are essential to the cell survival and have ability to prevent cardiac myocytes from detrimental outcomes in response to various stimuli. In addition, Bax and Bad expression integrates important functions that are related to apoptosis and facilitate the release of cytochrome c from mitochondria41. Caspases are a family of cysteine proteases that serve as key regulators in programmed cell death or apoptosis. Among them, caspase-3 frequently activates apoptosis for catalyzing the specific cleavage and activation of many effectors, including caspase-6, caspase-7, and SREBPs42. Thus, upregulation of Bcl-2 and Bcl-xL, downregulation of Bad and Bax, and inhibition of caspase-3 activity are regarded as the hallmarks of antiapoptotic ability. Here, we demonstrated that treatment with GLP-1(9–36) increased the expressions of Bcl-2 and Bcl-xL, whereas the expressions of Bad and Bax were not changed. In addition, GLP-1(9–36) reduced the expression of Bad and Bax induced by H2O2 in H9c2 cardiomyoblasts (Fig. 5). These findings indicated that GLP-1(9–36) provokes pro-survival signaling pathways in the normal and H2O2-treated conditions, leading to improved apoptosis and enhanced cell survival after oxidative stress in the heart.
As we known that GLP-1(9–36) was considered to be an inactive peptide, thus, it might have the other signaling for GLP-1(9–36)-mediated protective effects which is independent of GLP-1 receptor. For instance, GLP-1(9–36) provoked cardioprotective effects against MG-induced mitochondrial dysfunction12. The cardioprotective and vasodilatory effects of GLP-1(7–36) are still observed even in GLP-1R knockout mice, which implied the beneficial roles of GLP-1(9–36) in GLP-1R independent fashion11. Consistent with these studies, we demonstrated that the antioxidant and antiapoptotic effects of GLP-1(9–36) were independent of GLP-1R.
After exendin-4 binding to GLP-1R, this leads to the coupling and activation of Gαs protein by GLP-1R, resulting in an increase of cAMP levels through the activated AC. After that cAMP binds to and interacts with its effectors, including PKA and Epac. Elevation of cAMP levels by GLP-1R stimulation have been shown to have cardioprotective effects in mouse cardiomyocytes43 and H9c2 cardiomyoblasts12. Although treatment with GLP-1(9–36) increased cAMP levels, this effect of GLP-1(9–36) was less than that of exendin-4 (a potent GLP-1R agonist) (Fig. 2E). In addition, blockade of AC activity by DDA had no effect on GLP-1(9–36)-mediated antioxidant and antiapoptotic effects in H9c2 cardiomyoblasts, emphasizing cardioprotective effects of GLP-1(9–36) are not mediated through cAMP-signaling pathway. Here we demonstrated that GLP-1(9–36) exerts its effects through PI3K/Akt/NOS signaling pathway (Fig. 8).
The PI3K/Akt axis is an essential signaling pathway that promotes cell survival and is responsible for regulation of apoptosis19. The role of PI3K and Akt in the prevention of oxidative stress and apoptosis in cardiac cells has been described. Stimulation of Akt activity is capable of suppressing H2O2 induced apoptosis in cardiac myocytes44. Treatment with GLP-1 inhibited palmitate-induced apoptosis in cardiomyocytes, in which this protective effect was blocked by PI3K inhibitor45. Furthermore, treatment with exendin-4 provokes the activation of Akt activity in H9c2 cells12. Consistent with these studies, blockade of either PI3K activity or Akt activity was able to suppress GLP-1(9–36)-induced the synthesis of antioxidant enzymes and antiapoptotic proteins. GLP-1(9–36) effects on the inhibition of caspase-3 activity and apoptosis were also blocked in the presence of PI3K inhibitor or Akt inhibitor. Collectively, PI3K and Akt are necessary for GLP-1(9–36)-mediated antioxidant and antiapoptotic effects in the heart. We also reported here that GLP-1(9–36) induces the phosphorylation and activation of Akt in the PI3K-dependent way in H9c2 cardiomyoblast, emphasizing that PI3K and Akt act as downstream effectors for GLP-1(9–36) signaling. However, how GLP-1(93 − 6) regulate the activation of PI3K is not known and further study is needed to identify the mechanism for GLP-1(9–36)-mediated PI3K activation in the heart.
Nitric oxide synthase (NOS) is recognized as one of the important effectors of Akt in which Akt mediates phosphorylation and activation of NOS46,47. The important role of NOS in protecting the cells has been described and might be reflected on NO production in the cells48,49. Our results showed that blockade of NOS inhibited the cardioprotective effects of GLP-1(9–36) against H2O2, evidenced by a decrease in ROS production and caspase-3 activity. Moreover, depletion of NOS activity also attenuated GLP-1(9–36)-induced the upregulation of antioxidant enzymes (Fig. 6) and antiapoptotic proteins (Fig. 7) in H9c2 cardiomyoblasts.
In HUVECs, GLP-1 induced eNOS synthesis and activity via both the GLP-1R-dependent and GLP-1(9–36)-depenent manners22. GLP-1(9–36), but not exendin-4, protected the cells from exposure to I/R or H2O2 via the NOS-dependent way in human aortic endothelial cells isolated from GLP-1R knockout mice18. In addition, nNOS-mediated pathway has an essential role in protection of the heart from I/R injury50. Consistent with these previous studies, we demonstrated that GLP-1(9–36) increased the synthesis of nNOS and eNOS, leading to an increase in NO levels in H9c2 cardiomyoblasts. In contrast, GLP-1(9–36) had no effect on iNOS mRNA expression. Inhibition of PI3K/Akt pathway using specific PI3K and Akt inhibitors attenuated GLP-1(9–36)-mediated NO production. Thus, cardioprotective effects of GLP-1(9–36) were mainly related to upregulation of nNOS and eNOS through PI3K/Akt axis. Our data support a concept whereby a cleavage product of GLP-1, GLP-1(9–36) provokes cardioprotective effects in ex vivo and in vivo models of cardiac injury.
In conclusion, we have revealed novel insights into the molecular mechanisms of a truncated metabolite of GLP-1 hormone, GLP-1(9–36) for cardioprotective effects against oxidative stress and apoptosis (Fig. 8). GLP-1(9–36) exhibits antioxidant effects by reducing ROS production and enhancing the expressions of catalase, GPx-1, and HO-1, and exerts antiapoptotic effects by inhibiting caspase-3 activity and inducing Bcl-2 and Bcl-xL synthesis. These protective effects of GLP-1(9–36) are mediated through PI3K/Akt/NOS signaling pathway.