在細胞內自噬作用是一種降解錯誤合成蛋白的過程。許多器官在高糖的情況下，自噬作用扮演著保護的角色。在本實驗室先前體內和體外研究指出，高糖和糖尿病鼠會誘導心肌凋亡的現象。然而，自噬作用在高糖所誘導的細胞死亡中是否扮演著保護的角色則是未知的。利用H9c2心肌細胞，我們證明了33mM 葡萄糖濃度會誘導自噬作用的生物指標，Beclin-1, Atg5, LC3-II 的增加。利用免疫螢光檢測短暫轉殖GFP-LC3形成之亮點，也得到同樣結果。我們發現在TUNEL實驗結果中，3-甲基腺素-6-氨基-3-甲基嘌呤 (3-MA；自噬作用抑制劑) 會強化Caspase3的活性，雷帕埋辛 (Rapamycin；自噬作用促進劑) 會減少Caspase3的活性；暗示著自噬作用在高糖情況下扮演著保護的角色。更進一步，證明處理33mM 葡萄糖會增加腺苷酸活化蛋白激酶 (AMPK) 表現量，加入化合物C (Compound C；腺苷酸活化蛋白激酶抑制劑) 可強化自噬作用，5-氨基咪唑-4-甲醯胺基核苷酸轉甲醯基酶 (AICAR；腺苷酸活化蛋白激酶促進劑) 則會抑制自噬作用表現。我們的結果證明了，藉由化合物C可減緩 H9c2在高糖情形下所誘發的凋亡，5-氨基咪唑-4-甲醯胺基核苷酸轉甲醯基酶則會強化Caspase3的活性；在小分子干擾RNA (siRNA) 實驗中，小分子干擾腺苷酸活化蛋白激酶亞法1 (AMPKα1si) 可減緩高糖情形下所誘發的凋亡。暗示著處理在高糖的情形下自噬作用的活性是藉由腺苷酸活化蛋白激酶的調控，在H9c2心肌細胞中扮演著保護的角色。
Autophagy is a process of bulk degradation of intracellular components and shows protective effects in many organs under high glucose condition. Our previous in vivo and in vitro studies demonstrate that high glucose (HG) and diabetic rats can induce cardiac apoptosis. However, whether autohpagy plays a protective role on HG induced cardiac cell death is unknown. Using H9c2 cells, we demonstrate that autophagy biomarker Beclin-1, Atg5, LC3-II were increased following 33mM glucose treatment. The transfected GFP-LC3 detected by immunofluresence assay showed similar results. We also found that cell death from the results of caspase 3 levels and TUNEL assay was enhanced by 3-MA as autophagy inhibitor and was decreased by rapamycin as autophagy enhancer, indicating autophagy plays a protective role in H9c2 cells exposed to HG. Furthermore, we also demonstrated that phosphorylation of 5’ adenosine monophosphate-activated protein kinase (AMPK) were time dependent increased following 33mM glucose treatment, and the HG induced autophagy was enhanced by Compound C (CC) used as AMPK inhibitor and was inhibited by 5-aminoimidazole-4-carboxamide-β-D-riboside (AICAR) used as AMPK promoter. Our results also showed that stimulation of CC and AMPKα1si rescued H9c2 cells from HG induced cell death and administration of AICAR augmented caspase3 activation, suggesting that an increase of autophagy through modulation of AMPK plays a protective role on H9c2 cells exposed to HG
