Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes.
Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes.
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DOI：
10.7554/elife.38519
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发表时间：
2018-11-09
期刊：
影响因子：
7.7
通讯作者：
Otonkoski T
中科院分区：
文献类型：
--
作者：
Balboa D;Saarimäki-Vire J;Borshagovski D;Survila M;Lindholm P;Galli E;Eurola S;Ustinov J;Grym H;Huopio H;Partanen J;Wartiovaara K;Otonkoski T
胰岛素基因突变是新生儿糖尿病的主要原因。它们可导致胰岛素原错误折叠并滞留在内质网（ER）中。这导致ER应激增加，提示触发β细胞凋亡。在人类中，β细胞衰竭的潜在机制仍不清楚。在这里，我们表明，错误折叠的胰岛素原损害发展中的β细胞增殖，而不增加凋亡。我们从携带胰岛素（INS）突变的人中产生了诱导多能干细胞（iPSC），设计了等基因CRISPR-Cas9突变纠正系，并将它们分化为β样细胞。单细胞RNA测序分析显示，与校正对照相比，INS突变β样细胞的ER应激增加，增殖减少。在移植到小鼠中后，INS突变移植物呈现胰岛素分泌减少和ER应激加重。INS突变β样细胞的细胞大小、mTORC 1信号传导和呼吸链亚基表达均减少，但凋亡在任何阶段均未增加。我们的研究结果表明，新生儿糖尿病相关的INS突变导致有缺陷的β细胞群扩增，有助于糖尿病的发展。胰岛素是一种对维持正常血糖水平至关重要的激素，由胰腺中所谓的β细胞产生。如果体内的β细胞停止制造胰岛素，血糖水平开始上升，这可能导致糖尿病。一种被称为新生儿糖尿病的糖尿病，身体停止制造胰岛素，通常出现在生命的前六个月。受这种早期糖尿病影响的婴儿通常在编码胰岛素的基因的一个拷贝中发生突变。这意味着他们仍然可以产生一半的胰岛素，但不足以保持血糖稳定。相反，胰岛素生产在几个月后完全停止。科学家认为，这是因为突变胰岛素对β细胞有毒性作用。胰岛素基因的突变会影响胰岛素的结构。结果，胰岛素在β细胞内积累，这给它们带来压力，最终使它们衰竭。这一过程背后的机制尚不清楚。现在，Balboa等人使用从这种罕见的胰岛素突变患者身上提取的干细胞（可以转化为其他细胞类型）来发现更多。他们用一种名为CRISPR的技术纠正了这些干细胞中的突变胰岛素基因，然后诱导突变和纠正的干细胞变成β细胞。结果表明，突变的β细胞减慢了细胞分裂的速度，但并没有更频繁地死亡。当这些细胞被植入小鼠体内时，它们的生长和发育发生了变化。突变细胞比修复基因的细胞更受压力，更小。他们也有更少的信号分子，帮助细胞生长。因此，细胞正在努力生长和成熟。虽然这种类型的糖尿病很罕见，但β细胞在其他形式的疾病中会受到压力。在另一项研究中，Riahi等人发现，增强细胞生长的分子信号可以保护突变胰岛素小鼠的β细胞。如果这也能在人类身上发挥作用，它可能会导致预防糖尿病的新方法。
Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated induced pluripotent stem cells (iPSCs) from people carrying insulin (INS) mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation into mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunits expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development. Insulin is a hormone that is crucial for maintaining normal blood sugar levels and is produced by so called beta cells in the pancreas. If the beta cells in the body stop making insulin, blood sugar levels start to rise, which can lead to diabetes. A form of diabetes known as neonatal diabetes, where the body stops making insulin, usually appears during the first six months of life. Infants affected by this early onset of diabetes often have mutations in one copy of the gene that encodes insulin. This means that they can still produce half of the amount of insulin, but it is not enough to keep blood sugar stable. Instead, insulin production stops completely after a few months. Scientists believe that this is because the mutant insulin has a toxic effect on beta cells. Mutations in the insulin gene can affect the structure of insulin. As a result, insulin accumulates inside the beta cells, which stresses them and eventually makes them fail. The mechanisms behind this process are still unclear. Now, Balboa et al. used stem cells (which can turn into other cell types) taken from patients with this rare type of insulin mutation to find out more. They corrected the mutant insulin gene in these stem cells with a technique called CRISPR and then induced the mutant and corrected stem cells to turn into beta cells. The results showed that the mutant beta cells slowed down their rate of cell division but did not die more frequently. When the cells were implanted into mice their growth and development changed. The mutant cells were more stressed and smaller than the cells with the repaired genes. They also had fewer signalling molecules that help cells grow. As a consequence, the cells were struggling to grow and mature. Although this type of diabetes is rare, beta cells come under stress in other forms of the disease. In a separate study, Riahi et al. found that boosting molecular signals for cell growth could protect beta cells in mice with mutant insulin. If this could also work in humans, it may lead to new ways to prevent diabetes.