腸道葡萄糖授予缺氧上皮細胞抗死亡之 訊息傳遞路徑探討

Enteral glucose confers anti-death signaling in epithelial cells under hypoxic stress

黃菁英(Ching-Ying Huang)

指導教授 ： 余佳慧

國立臺灣大學/醫學院/生理學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00302

摘要

腸胃道受缺血或低氧壓力引發之上皮細胞死亡調節失常現象，與器官功能異常和腫瘤生成有關。先前的研究發現，腸道葡萄糖可透過未知機制授予腸道上皮細胞抗死亡之能力。活化第一型鈉-葡萄糖轉運蛋白(sodium/glucose transporter 1 ，SGLT1)可避免腸道上皮細胞因微生物刺激而走向細胞凋亡。除此之外，有文獻記載在人類大腸直腸癌細胞的壞死區域周圍，葡萄糖轉運蛋白(Glucose transporters, GLUTs) 的異常表現會伴隨低氧誘發因子(Hypoxia inducicle factor (HIF)1α)共存，推論葡萄糖攝取增加與缺氧區域之抗壞死有關。第一部份之研究目的，欲透過動物實驗探討腸腔中葡萄糖對腸繫膜缺血造成之上皮細胞死亡與腸道屏障功能失常之保護作用，並進一步研究葡萄糖所媒介之細胞生存訊息傳遞路徑。第二部分之研究目的，則利用人類大腸直腸癌細胞株，探討缺氧誘發細胞計畫性壞死之訊息傳遞路徑，以及葡萄糖代謝物丙酮酸在細胞死亡阻抗性中所扮演的角色。
在第一部份的實驗中發現，將上腸繫膜動脈夾閉造成缺血刺激，會快速引發空腸上皮細胞凋亡。而缺血後再灌流，則造成凋亡蛋白酶依賴性之腸道通透性上升，腸道細菌轉移增加，以及腸黏膜發炎。腸道葡萄糖經由SGLT1攝取，降低缺血再灌流引發之上皮細胞凋亡、屏障受損，以及黏膜發炎；其乃藉由活化抗凋亡之PI3K/Akt路徑及其下游訊息傳遞分子，包括磷酸化mTOR 與 Bad蛋白，去磷酸化p38 MAPK蛋白。
在第二部份的實驗中，將人類大腸直腸癌細胞暴露於缺乏氧氣與葡萄糖之環境中，可誘發其發生計劃性壞死，包括receptor-interacting protein kinase (RIP)依賴性乳酸去氫酶之滲漏與RIP1/3複合體生成。缺氧刺激後亦誘發粒線體中超氧化物產生；而給予抗氧化物之後則可抑制RIP訊息與細胞壞死。RIP依賴性之計畫性壞死，在給予葡萄糖後消失；但若將葡萄糖以其他非代謝性醣類似物取代則無此效果。缺氧細胞在給予葡萄糖後HIF1α有轉移細胞核的現象，且與GLUT1與GLUT4之表現增加具關聯性。醣解代謝授予缺氧癌細胞對計劃性壞死之阻抗性，乃部分源自於丙酮酸清除粒線體自由基的作用且不需依賴能量生成
總括而論，我們發現給予腸道葡萄糖可降低因缺氧壓力造成之細胞凋亡或計畫性壞死，其抗死亡之作用乃分別透過活化抗凋亡之PI3K/Akt訊息傳遞路徑；或是經由丙酮酸對粒線體自由基之清除作用。

關鍵字

葡萄糖 ； 腸道上皮細胞 ； 低氧壓力 ； 細胞凋亡 ； 細胞計劃性壞死

並列摘要

Dysregulated epithelial cell death caused by ischemic or hypoxic stress is associated with malfunction or tumorigenesis in gastrointestinal tract. Previous studies have shown that enteral glucose uptake confers resistance to epithelial cell death via unknown mechanisms. Activation of sodium/glucose transporter 1 (SGLT1) prevented epithelial apoptosis caused by microbial products. In addition, abnormally expressed glucose transporters (GLUTs) were found colocalized with hypoxia inducible factor (HIF)1α in peri-necrotic regions in human colorectal carcinoma, suggesting that enhanced glucose uptake may be linked with anti-necrotic resistance in hypoxic tumor core. The aim of the first study was to explore whether luminal glucose uptake protects against mesenteric ischemia-induced epithelial cell death and intestinal barrier dysfunction, and to explore the glucose-mediated cellular survival pathways in vivo. The aim of the second study is to investigate signaling pathways of hypoxia-induced necroptosis and explore the role of glucose pyruvate metabolite in mechanisms of death resistance in human colorectal cancer cells.
In the first part of study, ischemic challenge by occlusion of superior mesenteric artery caused rapid onset of enterocyte apoptosis in rat jejunum. Ischemia-reperfusion (I/R) triggered rise of intestinal permeability, augmentation of bacterial translocation (BT) and mucosal inflammation in a caspase-dependent manner. SGLT1-mediated enteral glucose attenuated I/R-induced epithelial apoptosis, barrier damage, and mucosal inflammation via activation of anti-apoptotic PI3K/Akt signaling, including phosphorylation of mTOR and Bad, and dephosphorylation of p38 MAPK.
In the second part of study, human colorectal carcinoma cells hypoxia exposed in glucose-free media displayed signs of necroptosis, including receptor interacting protein (RIP)-dependent lactodehydrogenase leakage and RIP-1/3 complex formation. Generation of mitochondrial superoxide was noted after hypoxic challenge; its reduction by antioxidants inhibited RIP signaling and cell necroptosis. Addition of glucose and pyruvate derivative, but not non-metabolizable analogs, diminished RIP-dependent necroptosis. Hypoxic cells with glucose showed HIF1α activation and hypoxia-targeted gene (GLUT-1 and -4) expression. Glycolytic pyruvate scavenging of hypoxia-induced mitochondrial superoxide was involved in anti-necrotic mechanisms in an energy-independent manner.
In conclusion, we found that enteral glucose uptake attenuated both type of cell death, apoptosis and necroptosis in gut epithelial cell under hypoxic stress via either anti-apoptotic PI3K/Akt signaling pathway or through pyruvate scavenging of mitochondrial free radical, respectively.

並列關鍵字

glucose ； intestinal epithelium ； hypoxic stress ； apoptosis ； necroptosis

參考文獻

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