Bensellam Mohammed, Maxwell Emma L, Chan Jeng Yie, Luzuriaga Jude, West Phillip K, Jonas Jean-Christophe, Gunton Jenny E, Laybutt D Ross
Garvan Institute of Medical Research, St Vincent's Hospital, UNSW Australia, 384 Victoria Street, Darlinghurst, Sydney, NSW, 2010, Australia.
Université catholique de Louvain, Institut de recherche expérimentale et clinique, Pôle d'endocrinologie, diabète et nutrition, Brussels, Belgium.
Diabetologia. 2016 Jul;59(7):1492-1502. doi: 10.1007/s00125-016-3947-y. Epub 2016 Apr 4.
AIMS/HYPOTHESIS: Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress.
Mouse islets and MIN6 cells were exposed to various oxygen (O2) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA; Hspa5 was also overexpressed. db/db mice were used.
Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic, db/db mice. In isolated mouse islets and MIN6 cells, O2 deprivation (1-5% vs 20%; 4-24 h) markedly reduced the expression of adaptive UPR genes, including Hspa5, Hsp90b1, Fkbp11 and spliced Xbp1. Coatomer protein complex genes (Copa, Cope, Copg [also known as Copg1], Copz1 and Copz2) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes (Ddit3, Atf3 and Trb3 [also known as Trib3]), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally, Hspa5 overexpression significantly protected against hypoxia-induced cell death.
CONCLUSIONS/INTERPRETATION: Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress.
目的/假设：缺氧可能导致2型糖尿病和胰岛移植中的β细胞功能衰竭。适应性未折叠蛋白反应（UPR）是内质网（ER）稳态所必需的。在此，我们研究了缺氧是否调节β细胞中的UPR以及适应性UPR在缺氧应激期间所起的作用。
将小鼠胰岛和MIN6细胞暴露于不同的氧（O2）张力下。使用小干扰（si）RNA敲低DNA损伤诱导转录物3（DDIT3）、缺氧诱导转录因子（HIF）1α和HSPA5；Hspa5也过表达。使用db/db小鼠。
在糖尿病db/db小鼠的胰岛中，缺氧反应基因在体内上调，但在糖尿病前期db/db小鼠中未上调。在分离的小鼠胰岛和MIN6细胞中，缺氧（1-5% 对20%；4-24小时）显著降低了适应性UPR基因的表达，包括Hspa5、Hsp90b1、Fkbp11和剪接的Xbp1。衣被蛋白复合物基因（Copa、Cope、Copg [也称为Copg1]、Copz1和Copz2）以及内质网到高尔基体的蛋白质转运也减少，而凋亡基因（Ddit3、Atf3和Trb3 [也称为Trib3]）、c-Jun氨基末端激酶（JNK）磷酸化和细胞死亡增加。抑制JNK而非HIF1α可恢复适应性UPR基因表达和内质网到高尔基体的蛋白质转运，同时在缺氧后保护细胞免受凋亡基因影响和细胞死亡。DDIT3敲低延迟了适应性UPR的丧失，并部分保护细胞免受缺氧诱导的细胞死亡。HSPA5敲低可阻止后一种反应。最后，Hspa5过表达显著保护细胞免受缺氧诱导的细胞死亡。
结论/解读：缺氧通过JNK和DDIT3激活抑制β细胞中的适应性UPR，但独立于HIF1α。适应性UPR的下调导致缺氧应激期间内质网到高尔基体的蛋白质转运减少和β细胞死亡增加。
