Accumulation of reactive oxygen species (i.e., oxidative stress) is a leading cause of beta cell dysfunction and apoptosis in diabetes. NRF2 (NF-E2 p45-related factor-2) regulates the adaptation to oxidative stress, and its activity is negatively regulated by the redox-sensitive CUL3 (cullin-3) ubiquitin ligase substrate adaptor KEAP1 (Kelch-like ECH-associated protein-1). Additionally, NRF2 is repressed by the insulin-regulated Glycogen Synthase Kinase-3 (GSK3). We have demonstrated that phosphorylation of NRF2 by GSK3 enhances β-TrCP (beta-transducin repeat-containing protein) binding and ubiquitylation by CUL1 (cullin-1), resulting in increased proteasomal degradation of NRF2. Thus, we hypothesise that inhibition of GSK3 activity or β-TrCP binding upregulates NRF2 and so protects beta cells against oxidative stress. We have found that treating the pancreatic beta cell line INS-1 832/13 with the KEAP1 inhibitor TBE31 significantly enhanced NRF2 protein levels. The presence of the GSK3 inhibitor CT99021 or the β-TrCP-NRF2 protein-protein interaction inhibitor PHAR, along with TBE31, resulted in prolonged NRF2 stability and enhanced nuclear localisation (P < 0.05). TBE31-mediated induction of NRF2-target genes encoding NAD(P)H quinone oxidoreductase 1 (), glutamate-cysteine ligase modifier () subunit and heme oxygenase () was significantly enhanced by the presence of CT99021 or PHAR (P < 0.05) in both INS-1 832/13 and in isolated mouse islets. Identical results were obtained using structurally distinct GSK3 inhibitors and inhibition of KEAP1 with sulforaphane. In summary, we demonstrate that GSK3 and β-TrCP/CUL1 regulate the proteasomal degradation of NRF2, enhancing the impact of KEAP1 regulation, and so contributes to the redox status of pancreatic beta cells. Inhibition of GSK3, or β-TrCP/CUL1 binding to NRF2 may represent a strategy to protect beta cells from oxidative stress.

中文翻译：

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抑制糖原合成酶激酶 3 可增强胰腺 β 细胞中 NRF2 蛋白的稳定性、核定位和靶基因转录


活性氧的积累（即氧化应激）是糖尿病中β细胞功能障碍和细胞凋亡的主要原因。 NRF2（NF-E2 p45 相关因子 2）调节对氧化应激的适应，其活性受到氧化还原敏感的 CUL3（cullin-3）泛素连接酶底物适配器 KEAP1（Kelch 样 ECH 相关蛋白-）的负调节。 1）。此外，NRF2 受到胰岛素调节的糖原合酶激酶 3 (GSK3) 的抑制。我们已经证明，GSK3 对 NRF2 的磷酸化增强了 β-TrCP（含β转导蛋白重复序列​​的蛋白）的结合和 CUL1 (cullin-1) 的泛素化，从而导致 NRF2 的蛋白酶体降解增加。因此，我们假设抑制 GSK3 活性或 β-TrCP 结合会上调 NRF2，从而保护 β 细胞免受氧化应激。我们发现用 KEAP1 抑制剂 TBE31 处理胰腺 β 细胞系 INS-1 832/13 显着增强 NRF2 蛋白水平。 GSK3 抑制剂 CT99021 或 β-TrCP-NRF2 蛋白质-蛋白质相互作用抑制剂 PHAR 以及 TBE31 的存在，导致 NRF2 稳定性延长并增强核定位 (P < 0.05)。 CT99021 或 PHAR 的存在显着增强了 TBE31 介导的编码 NAD(P)H 醌氧化还原酶 1 ()、谷氨酸半胱氨酸连接酶修饰剂 () 亚基和血红素加氧酶 () 的 NRF2 靶基因的诱导 (P < 0.05) INS-1 832/13 和分离的小鼠胰岛中。使用结构不同的 GSK3 抑制剂和用萝卜硫素抑制 KEAP1 获得了相同的结果。总之，我们证明 GSK3 和 β-TrCP/CUL1 调节 NRF2 的蛋白酶体降解，增强 KEAP1 调节的影响，从而有助于胰腺 β 细胞的氧化还原状态。 抑制 GSK3 或 β-TrCP/CUL1 与 NRF2 的结合可能是保护 β 细胞免受氧化应激的策略。