DPP4基因缺陷產生心臟保護作用之機轉探討

Abstract

攝食會刺激GLP-1分泌降低血糖，但DPP4酵素很快的將GLP-1切斷，使其失去調控血糖功能。抑制DPP4酵素活性可提升血液中GLP-1含量，所以在臨床上DPP4抑制劑及GLP-1類似物已用於治療糖尿病。除了調控血糖外，GLP-1訊息也被發現能夠改善心血管系統相關疾病，所以我們以動物及細胞實驗模擬人類疾病，探討DPP4基因突變是否影響心血管功能。 以成年大鼠實驗：wild-type為野生種，DchcHsd-DPP IV為DPP4基因缺陷大鼠。DPP4活性在DPP4基因缺陷大鼠中只有野生種的三分之一。動物實驗是比較兩種動物面臨內毒素血症及心肌梗塞的耐受性。心臟功能是以心導管偵測心臟壓力及體積來評估。為了探討GLP-1訊息傳遞路徑，有些組別中使用GLP-1類似物（exendin-4）或其受器阻斷劑（exendin-(9-39)）。動物血清及心臟組織在模擬疾病實驗結束後收集儲存。細胞實驗是分離兩種大鼠心臟細胞，並探討細胞對抗H2O2產生氧化壓力的能力，以細胞存活量、胞內自由基含量及細胞凋零相關路徑活化作為評估。 以靜脈注射10mg/kg內毒素誘導內毒素血症，模擬敗血症的大量發炎反應。所有實驗都在施打四小時後進行分析。在野生種大鼠中，內毒素造成心臟功能減弱，這與減少心臟中cAMP含量、phospholamban磷酸化及減弱血管對交感神經興奮劑反應有關。DPP4基因缺陷能改善內毒素血症時心血管功能，這與動物體內產生較多GLP-1有關，因為這結果與外給GLP-1類似物（exendin-4）的保護作用相符合：在野生種大鼠施打exendin-4能夠有效改善內毒素血症所引起的心血管功能降低現象。而且DPP4基因缺陷或是GLP-1類似物亦能改善內毒素血症造成的多重器官衰竭及降低死亡率。總之，這個實驗證明了在面臨內毒素血症時DPP4基因缺陷能使血液中分泌高含量的GLP-1以達保護效果。 在心臟梗塞的動物實驗中，大鼠冠狀動脈綑綁45分鐘接續鬆開兩小時，模擬心臟缺血再灌流。與野生種大鼠相比，DPP4大鼠在面臨心臟缺血再灌流後能維持較佳的作功能力，和較少的梗塞壞死面積，並伴隨心臟受損相關生化數值降低（LDH、ANP和BNP）；而GLP-1受器拮抗劑（exendin-(9–39)）會減弱上述保護效果，並減少心臟缺血再灌流後AKT及其下游蛋白GSK-3β磷酸化及GLUT4表現量。然而特別的是：給予DPP4大鼠 exendin-(9–39)，雖會使心肌梗塞面積升高，但相較於野生種大鼠仍具有保護作用，這意味著與GLP-1受器無關路徑也參與其中。因此由這實驗可得知，DPP4基因缺陷對於心肌梗塞具有保護作用，這作用與GLP-1受器依賴路徑有關或無關。 DPP4基因缺陷在內毒素血症及心臟缺血再灌流都具保護作用，但機轉是因為單純的降低DPP4活性而放大GLP-1訊息，還是降低DPP4活性導致功能上改變而誘導新的路徑，並未釐清。所以接下來的實驗，是分離兩種大鼠心臟細胞，探討其對抗H2O2所造成的氧化壓力能力。 心臟細胞由兩種成年大鼠中分離，以H2O2處理，並外給GLP-1探討其中機轉。DPP4基因缺陷會減少H2O2誘導的胞內自由基產生、Bax/Bcl2比例及caspase-3活性，並改善H2O2所造成的細胞死亡。外給GLP-1於野生種心臟細胞能藉由磷酸化AKT，有效減少H2O2造成的細胞死亡；並且這保護作用完全被GLP-1受器拮抗劑阻擋，顯示在此GLP-1依存路徑的重要性。但是DPP4基因缺陷的心臟細胞面臨氧化壓力時，會增加AKT磷酸化，而外加GLP-1並沒有再額外增加磷酸化，顯示在DPP4基因缺陷時會另外活化與GLP-1無關的保護路徑。 由上述這幾個實驗證實：DPP4基因缺陷會導致體內含有較高濃度的GLP-1，能有效對抗內毒素血症及心肌梗塞所造成的心臟功能降低。這保護機制與GLP-1受器依賴作用相關或無關。其中GLP-1受器依賴作用是經由磷酸化AKT來降低心臟損傷。DPP4抑制劑與GLP-1類似物除了能調控血糖外，對於糖尿病患心血管系統有相當好的保護作用，甚至能作為敗血症及心肌梗塞疾病新型預防或治療用藥。

Dipeptidyl peptidase-4 (DPP4) enzyme inhibition has been reported to increase plasma glucagon-like peptide-1 (GLP-1) level for controlling postprandial glucose concentration. Both DPP4 inhibitors and GLP-1 analogue have been approved as antihyperglycemic agents in the treatment of diabetes. In addition to the insulinotropic effect, GLP-1 signaling was discovered for the improvement of cardiovascular disease. We examined whether genetic mutation of DPP4 influence cardiac response in both animal model experiments and cell studies. Adult Fischer 344 (wild type) and DchcHsd-DPP IV (served as DPP4 deficiency) rats were used. DPP4 activity of DPP4-deficient rats is about one third of that in wild-type rats. Animal model experiments of endotoxemia and myocardial infarction were performed and compared in two kinds of rats. Cardiac function was assessed by pressure-volume loop monitoring. In some studies, GLP-1 analogue (exendin-4) or receptor antagonist (exendin-(9-39)) were used to identify the signaling of the protective effect. The blood plasma samples were collected and the hearts were harvested at the end of the experimental model. Adult cardiomyocyte was also isolated in two kinds of rats, and the effects of H2O2 induced ROS stress were compared. Cell viability, ROS staining, and proapoptotic signaling were executed to determine the response to H2O2. Endotoxemia was induced by the administration of lipopolysaccharide (LPS, 10 mg/kg, i.v.), and all the experiments were performed after 4 h of treatment. LPS-induced suppression of cardiovascular function in wild-type rats was associated with a significant reduction in cardiac cAMP level, phosphorylation of phospholamban, and attenuation of aortic contractile response to phenylephrine. DPP4-deficient rats had better preservation of cardiovascular function than wild-type rats during endotoxemia, which was correlated with a more prominent elevation of GLP-1 signaling. These findings coincided with the pretreatment of GLP-1 analogue, exendin-4, where the deterioration of cardiovascular function during endotoxemia was significantly reversed in wild-type rats. Furthermore, the benefit of DPP4 deficiency or GLP-1 analogue not only preserved cardiovascular function but also alleviated multiple organ injury and improved survival rate during endotoxemia. In brief, this study demonstrated that the resistance to LPS in DPP4-deficient rats seems to be derived from the higher GLP-1 production, while exendin-4 exerts protective effect in endotoxemia. For the myocardial infarction experimental models, rats were subjected to 45 min of coronary artery occlusion, and followed by reperfusion for 2 h. As compared to wild-type rats, after ischemia/reperfusion (I/R), DPP4-deficient rats had better cardiac performance in association with less infarct size and cardiac injury markers (LDH, ANP, and BNP), which could be attenuated by exendin-(9–39), a GLP-1 receptor antagonist. Exendin-(9–39) could diminish the increased phosphorylation levels of myocardial AKT and GSK-3β as well as the higher expression of GLUT4 in post-infarcted DPP4-deficient rats. However, exendin-(9–39) could not completely abrogate the less infarct size in DPP4-deficient rats as compared with that in wild-type rats, implicating the involvement of GLP-1 receptor-independent pathway. Accordingly, this study demonstrated that the benefit of cardiac protective action against I/R injury in DPP4-deficient rats, which is mediated through both GLP-1 receptor-dependent and receptor-independent mechanisms. DPP4-deficient rats show resistance to endotoxemia and ischemia/reperfusion. However, the decrease of DPP4 activity simply augmented the GLP-1 signaling or that such decrease resulted in a functional change or induced new signaling pathways remain unclear. With above reasons, we compared two kinds of cardiomyocytes under oxidative stress induced by H2O2. Cardiomyocytes were isolated from two kinds of rats. The effect of H2O2-induced ROS stress was performed in the presence or absence of GLP-1. DPP4-deficient cardiomyocytes were found to be resistant to H2O2-induced cell death via diminishing ROS level, Bax/Bcl-2 ratio, and caspase-3 activity. GLP-1 was also shown to decrease H2O2-induced cell death in wild-type cardiomyocytes via increasing the phosphorylation of AKT, which was abolished by exendin-(9–39), suggesting the importance of GLP-1 receptor dependent pathway. However, GLP-1 did not further increase phosphorylation of AKT against H2O2-induced stress in DPP4-deficient cardiomyocyte, indicating a crucial role of GLP-1-independent mechanism in these events. These several studies suggest that higher GLP-1 concentration in DPP4 mutant rats may contribute to the resistance to LPS and myocardial infarction. The protective effect is associated with both GLP-1 receptor -dependent and -independent pathway. The receptor dependent pathway is via increasing phosphorylation of AKT to ameliorate cardiac injury. In addition to the blood glucose controlling effect, GLP-1 receptor agonist or DPP4 inhibitor may exert cardioprotective effect in diabetes, and possibly be used as a preventive or even as a novel therapeutic agent in septic shock and myocardial infarction.