高醣終產物-羧甲基離氨基酸引發胰臟β細胞粒線體功能損傷及細胞死亡

Induced mitochondrial dysfunction and cell death of pancreatic β cells by Nε-(carboxymethyl) lysine

呂靜宜(Chin-I Lu)

指導教授 ： 高淑慧

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2007年)

摘要

糖尿病患長期處於高血糖的環境下，容易引發葡萄糖自動氧化 (autoxidation)與蛋白質醣化 (glycation)。在糖尿病及老化的過程中，高度醣化終產物advanced glycation end products (AGEs) 被證實其扮演了抑制細胞生長、分化及機能的角色。AGEs是蛋白質或脂質經過非酵素參與之醣化作用 (non-enzymatic glycosylation) 及氧化作用，所形成的最終物質。以羧甲基離氨基酸(Nε-(carboxymethyl) lysine (CML)) 及羧乙基離氨基酸 (carboxyethyl lysine (CEL))為存在於生物體內的主要形式。當AGEs在人體組織內累積過量時，會產生較多的reactive oxygen species (ROS)，使得體內氧化壓力增加，進而促使脂質過氧化物增加，並促使血中脂蛋白的醣化及氧化，往往是造成罹患糖尿病、慢性腎病變、動脈硬化症等重要的因素。beta細胞是人體內主要分泌胰島素的場所，研究指出當粒線體功能缺損，ATP形成不足會影響胰島素的分泌。本研究的主旨為探討CML是否經由引發粒線體功能缺損而影響beta細胞功能進而降低分泌胰島素的功能。本實驗是利用大鼠胰島beta細胞 (RIN-m5F細胞株) 為細胞模式，由結果顯示當beta細胞經6 μM CML處理24小時後，發現造成細胞存活率明顯下降到70.4% ± 0.8% (p < 0.001)，ROS的產量比對照組增加29.9% ± 15.9% (p < 0.01)。進一步分析粒線體膜電位，發現CML處理可降低粒線體膜電位至13.9% ± 2.3% (p < 0.001)。β細胞經CML處理6小時後，分析細胞內ATP含量，ATP含量減少到對照組的55.9% ± 10.5% (p < 0.01)。此外，我們也發現CML會產生脂質過氧化及粒線體基因重組突變。此外，β細胞的胰島素釋放減少至69.1% ± 0.5% ( p < 0.001)。當以西方墨點法分析發現CML處理可以使粒線體內UCP2增加至112.4% ± 0.1%，而UCP2的增加可能與粒線體的氧化磷酸化作用及膜電位降低有關。本研究發現，CML造成粒線體功能缺損進而降低β細胞的胰島素的含量及分泌能力。我們推論CML可能在β細胞功能缺損，及β細胞mass減少扮演重要的角色。

關鍵字

高醣終產物 ； 糖尿病 ； beta細胞

並列摘要

Abstract
Diabetes is caused by progressive β cell dysfunction, insulin secretion deficiency and insulin resistance. Advanced glycation end products (AGEs) are nonenzymatically formed by reducing glucose, lipids, and/or certain amino acids on proteins, lipids, and nucleic acids. Nε-(carboxymethyl) lysine (CML) and carboxyethyl lysine (CEL) modified proteins have been identified as major AGEs. In this study, we investigated whethen CML might cause β cell malfunction via mitochondrial dysfunction. We examined the effects of CML-BSA on cell viability, insulin synthesis and secretion, and mitochondrial function in rat pancreatic β cells (RIN-m5F cells). Treatment of RIN-M5F cells with CML-BSA (6 μM) reduced the cell viability 70.4% ± 0.8% (p < 0.001) by dye exclusion assay. CML-BSA increased ROS generation as demonstrated in a time-dependent manner. Treatment of cells with CML-BSA (6 μM) reduced ATP production, mitochondrial membrane potential, and insulin secretion by 55.9% ± 10.5% (p < 0.01), 13.9% ± 2.3% (p < 0.001), 69.1% ± 0.5% (p < 0.001) respectively. Furthermore, 12.4% ± 0.1% increased of UCP2 were found in the CML-BSA treated cells. The increased UCP might contribute the declined mitochondrial respiratory activity and mitochondrial membrane potential. These results suggest that CML could attenuate insulin secretion via mitochondrial dysfunction of β cells. CML-BSA might play an important role in the progressive β cells dysfunction and loss of β cells.

並列關鍵字

advanced glycation end production ； diabetes ； beta cell

參考文獻

Yan, S. D., Schmidt, A. M., Anderson, G. M., Zhang, J., Brett, J., Zou, Y. S., Pinsky, D., and Stern, D. (1994). Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem 269, 9889-9897.

Ahmed, N. (2005). Advanced glycation endproducts--role in pathology of diabetic complications. Diabetes Res Clin Pract 67, 3-21.

Alarcon, C., Wicksteed, B., Prentki, M., Corkey, B. E., and Rhodes, C. J. (2002). Succinate is a preferential metabolic stimulus-coupling signal for glucose-induced proinsulin biosynthesis translation. Diabetes 51, 2496-2504.

Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 90, 7915-7922.

Anello, M., Lupi, R., Spampinato, D., Piro, S., Masini, M., Boggi, U., Del Prato, S., Rabuazzo, A. M., Purrello, F., and Marchetti, P. (2005). Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients. Diabetologia 48, 282-289.