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Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells

Wang Jing, WANG Jin, WANG Juan-Juan, ZHANG Wei-Fang, JIAO Xiang-Ying

Department of Physiology, Shanxi Medical University, Taiyuan 030001, China

Abstract

Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

Key words: INS-1 islet cells; thioredoxin interacting protein ; autophagy ; apoptosis

Received: 2016-11-28　　Accepted: 2017-01-11

Corresponding author: 焦向英　　E-mail: jiaoxy@gmail.com

Citing This Article：

Wang Jing, WANG Jin, WANG Juan-Juan, ZHANG Wei-Fang, JIAO Xiang-Ying. Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells. Acta Physiol Sin 2017; 69 (4): 445-451 (in Chinese with English abstract).