Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Republic of Singapore.
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
Diabetologia. 2021 Nov;64(11):2534-2549. doi: 10.1007/s00125-021-05530-3. Epub 2021 Aug 27.
AIMS/HYPOTHESIS: We studied the effects of heterozygous human INS gene mutations on insulin secretion, endoplasmic reticulum (ER) stress and other mechanisms in both MIN6 and human induced pluripotent stem cells (hiPSC)-derived beta-like cells, as well as the effects of prolonged overexpression of mutant human INS in MIN6 cells.
We modelled the structure of mutant C109Y and G32V proinsulin computationally to examine the in silico effects. We then overexpressed either wild-type (WT), mutant (C109Y or G32V), or both WT and mutant human preproinsulin in MIN6 cells, both transiently and stably over several weeks. We measured the levels of human and rodent insulin secreted, and examined the transcript and protein levels of several ER stress and apoptotic markers. We also reprogrammed human donor fibroblasts heterozygous for the C109Y mutation into hiPSCs and differentiated these into pancreatic beta-like cells, which were subjected to single-cell RNA-sequencing and transcript and protein analyses for ER stress and apoptotic markers.
The computational modelling studies, and short-term and long-term expression studies in beta cells, revealed the presence of ER stress, organelle changes and insulin processing defects, resulting in a decreased amount of insulin secreted but not the ability to secrete insulin. By 9 weeks of expression of mutant human INS, dominant-negative effects of mutant INS were evident and beta cell insulin secretory capacity declined. INS patient-derived beta-like cells and single-cell RNA-sequencing analyses then revealed compensatory upregulation in genes involved in insulin secretion, processing and inflammatory response.
CONCLUSIONS/INTERPRETATION: The results provide deeper insights into the mechanisms of beta cell failure during INS mutation-mediated diabetes disease progression. Decreasing spliced X-box binding protein 1 (sXBP1) or inflammatory response could be avenues to restore the function of the remaining WT INS allele.
目的/假设：我们研究了杂合人 INS 基因突变对 MIN6 和人诱导多能干细胞（hiPSC）衍生的β样细胞中胰岛素分泌、内质网（ER）应激和其他机制的影响，以及突变型人 INS 在 MIN6 细胞中过表达时间延长的影响。
我们通过计算建模研究了突变型 C109Y 和 G32V 胰岛素的结构，以检查计算机模拟的影响。然后，我们在 MIN6 细胞中瞬时和稳定过表达野生型（WT）、突变型（C109Y 或 G32V）或 WT 和突变型人前胰岛素，持续数周。我们测量了人胰岛素和鼠胰岛素的分泌水平，并检测了几种 ER 应激和凋亡标志物的转录和蛋白水平。我们还将携带 C109Y 突变的异合子人供体细胞重编程为 hiPSC，并将其分化为胰岛β样细胞，对这些细胞进行单细胞 RNA 测序和 ER 应激及凋亡标志物的转录和蛋白分析。
计算机建模研究以及在β细胞中的短期和长期表达研究表明，存在 ER 应激、细胞器变化和胰岛素加工缺陷，导致胰岛素分泌量减少，但不影响胰岛素分泌能力。在表达突变型人 INS 9 周后，突变型 INS 的显性负效应明显，β细胞胰岛素分泌能力下降。随后，INS 患者来源的β样细胞和单细胞 RNA 测序分析显示，参与胰岛素分泌、加工和炎症反应的基因代偿性上调。
结论/解释：研究结果为 INS 突变介导的糖尿病疾病进展过程中β细胞衰竭的机制提供了更深入的了解。减少剪接 X 盒结合蛋白 1（sXBP1）或炎症反应可能是恢复剩余 WT INS 等位基因功能的途径。
