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Mechanism of 2-deoxy-D-ribose-induced β-cell damage : The cystine/glutamate antiporters system c- protects β-cell from oxidative stress

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Abstract
Pancreatic β-cells are vulnerable to oxidative stress, which is related to β-cell failure in type 2 diabetes. System c- is a sodium-independent, cystine/glutamate antiporter that mediates the exchange of extracellular L-cystine and intracellular L-glutamate. The import of L-cystine through this transporter is the limiting state of intracellular glutathione (GSH) synthesis that has a significant role in defending against oxidative stress. Our previous study reported that 2‑deoxy‑D‑ribose (dRib) induces oxidative damage through GSH depletion in pancreatic β cells. This study aims to elucidate the mechanism underlying oxidative stress-induced β-cell damage. We measured intracellular L-[14C]cystine uptake, GSH content, reactive oxygen species (ROS) levels, cytotoxicity, and apoptosis in the rat insulinoma cell line, RINm5F. Stimulation with dRib dose- and time-dependently decreased intracellular L-[14C]cystine uptake and GSH content and increased intracellular ROS levels, cytotoxicity, and apoptosis. The addition of 2-mercaptoethanol (2-ME), a cystine uptake enhancer, Na+-independently recovered the dRib-induced decreases in L-[14C]cystine uptake, GSH content, and cell viability. To determine whether system c- deficiency mediates the oxidative stress-induced β cell damage, we overexpressed xCT, the substrate-specific subunit of the system c-, using a lentiviral vector in RINm5F cells. The overexpression of xCT fully recovered the dRibinduced decreases in L-[14C]cystine uptake and GSH content and prevented the dRib-induced ROS rises, cytotoxicity, and apoptosis. In conclusion, the overexpression of xCT showed protective effects against dRib-induced oxidative damage in RINm5F cells. This result suggests that system c- deficiency plays a critical role in oxidative stress-induced β-cell damage.
Author(s)
Yoo, So yeon
Issued Date
2020
Awarded Date
2020. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009411
Affiliation
제주대학교 대학원
Department
대학원 의학과
Advisor
Koh, Gwan Pyo
Table Of Contents
1. Introduction 1
1.1 Role of hyperglycemia induced oxidative stress in β-cell dysfunction in type 2 diabetes 1
1.2 Redox regulation . 3
1.2.1 Glutathione system 4
1.2.2 The cysteine/cystine cycle and transporter 6
1.2.3 System c-: sodium-independent cystine/glutamate antiporter . 8
1.3 2-deoxy- D-ribose (dRib) induced pancreatic β-cell damage 10
1.3.1 2-deoxy-D-ribose (dRib) 10
1.3.2 dRib induced oxidative stress and apoptosis in β-cell . 11
1.4 Objectives 16
2. Materials and Methods 17
2.1 Materials 17
2.2 Cell cultures 18
2.3 Assessment of cell viability 18
2.4 Flow cytometry to assess apoptosis 19
2.5 Assessment of the intracellular levels of ROS 19
2.6 Determination of the intracellular levels of glutathione . 20
2.7 Measurement of cystine transport . 21
2.8 Cell transfection 22
2.9 RNA isolation and reverse transcription quantitative polymerase chain reaction (RT qPCR). 22
2.10 Statistical analysis. 23
3. Results . 24
3.1 dRib inhibits cystine transport and decreases intracellular GSH content . 24
3.2 2-ME prevents dRib-induced decreases in cystine transport, GSH content, and cell viability 27
3.3 2-ME Na+-independently prevents dRib-induced decreases in cystine transport . 31
3.4 SASP and HCA reduce cystine transport and GSH concentration, and the reductions are prevented by 2-ME 33
3.5 Enforced expression of xCT overcomes dRib-induced decreases in cystine transport, GSH content, and cell viability . 36
3.6 The enforced expression of xCT recovers the dRib-induced increase in intracellular levels of ROS and apoptosis 45
4. Discussion . 55
5. Conclusion 57
6. References . 58
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Yoo, So yeon. (2020). Mechanism of 2-deoxy-D-ribose-induced β-cell damage : The cystine/glutamate antiporters system c- protects β-cell from oxidative stress
Appears in Collections:
General Graduate School > Medicine
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