Department of Internal Medicine, Jeju National University School of Medicine, 15 Aran 13-gil, Jeju, 63241, Republic of Korea; Department of Internal Medicine, Jeju National University Hospital, 15 Aran 13-gil, Jeju, 63241, Republic of Korea.
Department of Internal Medicine, Jeju National University School of Medicine, 15 Aran 13-gil, Jeju, 63241, Republic of Korea.
Free Radic Biol Med. 2020 Jun;153:17-25. doi: 10.1016/j.freeradbiomed.2020.04.012. Epub 2020 Apr 16.
Pancreatic β-cells are vulnerable to oxidative stress, which promotes β-cell failure in type 2 diabetes. System χ 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 rate-limiting step in the glutathione (GSH) biosynthesis pathway that plays a significant role in antioxidative defense. Previously, we reported that 2-deoxy-d-ribose (dRib) induces oxidative damage through GSH depletion in pancreatic β-cells. In the current study, we elucidated the mechanism underlying the oxidative stress-induced β-cell damage. We measured the intracellular l-[C]cystine uptake, GSH content, reactive oxygen species (ROS) levels, cytotoxicity, and apoptosis in rat insulinoma cell line, RINm5F. Treatment of dRib decreased the intracellular l-[C]cystine uptake and GSH content and increased the intracellular ROS levels, cytotoxicity, and apoptosis in a time- and dose-dependent manner. Conversely, 2-mercaptoethanol (2-ME), a cystine uptake enhancer, recovered the dRib-induced decrease in l-[C]cystine uptake, GSH content, and cell viability in a Na-independent manner. In the case of isolated islets, dRib dose-dependently decreased the intracellular l-[C]cystine uptake and cell viability; however, dRib-induced cytotoxicity was completely recovered by adding N-acetyl cysteine (NAC). To confirm that system χ mediates the oxidative stress-induced β-cell damage, we overexpressed xCT (the substrate-specific subunit of system χ) using a lentiviral vector in RINm5F cells. Overexpression of xCT fully recovered the dRib-induced decrease in l-[C]cystine uptake and GSH content and prevented the dRib-induced increase in ROS levels, cytotoxicity, and apoptosis. The overexpression of xCT showed a protective effect against dRib-induced oxidative damage in RINm5F cells. Our study showed that dRib depletes intracellular GSH content through inhibition of cystine transport via system χ in β-cells.
胰岛β细胞易受氧化应激影响，这会导致 2 型糖尿病中β细胞衰竭。系统 χ 是一种不依赖于钠的胱氨酸/谷氨酸反向转运体，介导细胞外 l-胱氨酸和细胞内 l-谷氨酸的交换。通过该转运体摄取 l-胱氨酸是谷胱甘肽（GSH）生物合成途径中的限速步骤，该途径在抗氧化防御中起重要作用。先前，我们报道 2-脱氧-d-核糖（dRib）通过耗尽胰岛β细胞中的 GSH 诱导氧化损伤。在本研究中，我们阐明了氧化应激诱导的β细胞损伤的机制。我们测量了大鼠胰岛素瘤细胞系 RINm5F 中的细胞内 l-[C]胱氨酸摄取、GSH 含量、活性氧（ROS）水平、细胞毒性和细胞凋亡。dRib 处理以时间和剂量依赖的方式降低细胞内 l-[C]胱氨酸摄取和 GSH 含量，并增加细胞内 ROS 水平、细胞毒性和细胞凋亡。相反，胱氨酸摄取增强剂 2-巯基乙醇（2-ME）以不依赖于钠的方式恢复了 dRib 诱导的 l-[C]胱氨酸摄取、GSH 含量和细胞活力的降低。对于分离的胰岛，dRib 剂量依赖性地降低细胞内 l-[C]胱氨酸摄取和细胞活力；然而，添加 N-乙酰半胱氨酸（NAC）完全恢复了 dRib 诱导的细胞毒性。为了证实系统 χ 介导氧化应激诱导的β细胞损伤，我们使用慢病毒载体在 RINm5F 细胞中转染 xCT（系统 χ 的底物特异性亚基）。xCT 的过表达完全恢复了 dRib 诱导的 l-[C]胱氨酸摄取和 GSH 含量的降低，并防止了 dRib 诱导的 ROS 水平、细胞毒性和细胞凋亡的增加。xCT 的过表达对 RINm5F 细胞中的 dRib 诱导的氧化损伤具有保护作用。我们的研究表明，dRib 通过抑制β细胞中通过系统 χ 的胱氨酸转运来耗尽细胞内 GSH 含量。
