Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction,Diabetologia

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Progressive endoplasmic reticulum stress over time due to human insulin gene mutation contributes to pancreatic beta cell dysfunction
Diabetologia ( IF 8.4 ) Pub Date : 2021-08-27 , DOI: 10.1007/s00125-021-05530-3
Nur Shabrina Amirruddin _{1,

2} , Wei Xuan Tan _{1,

2} , Yaw Sing Tan ₃ , Daphne Su-Lyn Gardner ₄ , Yong Mong Bee ₄ , Chandra Shekhar Verma _{3,

5,

6} , Shawn Hoon ₇ , Kok Onn Lee ₂ , Adrian Kee Keong Teo _{1,

2,

8}

Affiliation

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.

Methods

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.

Results

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^+/C109Y 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.

Graphical abstract

中文翻译：

由于人胰岛素基因突变导致的进行性内质网应激导致胰腺β细胞功能障碍

目标/假设

我们研究了杂合人类INS基因突变对 MIN6 和人类诱导多能干细胞 (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，以及长期过表达的影响MIN6 细胞中的突变人类INS。

方法

我们通过计算模拟了突变 C109Y 和 G32V 胰岛素原的结构，以检查计算机模拟效应。然后，我们在 MIN6 细胞中过表达野生型 (WT)、突变型 (C109Y 或 G32V) 或 WT 和突变型人前胰岛素原，在数周内均短暂且稳定地过表达。我们测量了人类和啮齿动物分泌的胰岛素水平，并检查了几种 ER 应激和凋亡标志物的转录物和蛋白质水平。我们还将 C109Y 突变杂合子的人类供体成纤维细胞重编程为 hiPSC，并将其分化为胰腺 β 样细胞，对这些细胞进行单细胞 RNA 测序以及 ER 应激和凋亡标志物的转录和蛋白质分析。

结果

计算模型研究以及 β 细胞中的短期和长期表达研究揭示了 ER 应激、细胞器变化和胰岛素加工缺陷的存在，导致胰岛素分泌量减少，但不能分泌胰岛素。突变人类INS表达 9 周时，突变INS的显性负效应明显，β 细胞胰岛素分泌能力下降。INS ^+/C109Y患者来源的 β 样细胞和单细胞 RNA 测序分析随后揭示了参与胰岛素分泌、加工和炎症反应的基因的代偿性上调。