Aims/hypothesis

Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected ‘hub’ cells, important for the propagation of intercellular Ca²⁺ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion.

Methods

Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca²⁺ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging.

Results

Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT⁺ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. ‘Hub’ cells were less abundant in the NNAT⁺ population, consistent with epigenetic control of this functional specialisation.

Conclusions/interpretation

These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Graphical Abstract

中文翻译：

---

Nnat 上的差异 CpG 甲基化在 β 细胞异质性早期建立中的作用

目标/假设

胰岛内的β细胞代表异质群体，其中各个细胞亚群对胰岛素分泌的总体控制做出不同的贡献。其中包括高度连接的“枢纽”细胞亚群，对于细胞间 Ca ²⁺波的传播非常重要。功能性亚群也在人类 β 细胞中得到证实，其中亚型分布的改变在 2 型糖尿病中很明显。目前，人们对β细胞层次结构建立的分子机制知之甚少。表观基因组水平的变化提供了一种这样的可能性，我们在这里通过关注印记基因Nnat（编码神经元 [NNAT]）来探索这种可能性，该基因是正常胰岛素合成和分泌所必需的。

方法

使用在 R 下运行的 Seurat 4.0 和 ClusterProfiler 检查单细胞 RNA-seq 数据集。生成在Nnat增强子/启动子区域控制下表达增强 GFP 的转基因小鼠，用于 β 细胞的 FACS 和通过亚硫酸氢盐测序进行 CpG 甲基化的下游分析和分别是RNA测序。使用从胰腺祖细胞阶段删除从头甲基转移酶 DNA 甲基转移酶 3 α (DNMT3A) 的动物来探索启动子甲基化的控制。使用亲和纯化质谱法进行蛋白质组学，并通过 Cal-520 AM 和 Cal-590 AM 的快速共焦成像探索Ca ²⁺动力学。使用均质时间分辨荧光成像测量胰岛素分泌。

结果

Nnat mRNA 在离散的 β 细胞群中以发育阶段和 DNA 甲基化 (DNMT3A) 依赖性方式差异表达。因此，胚胎数据集的伪时间分析证明了胚胎发生过程中Nnat阳性和阴性亚群的早期建立。 NNAT 表达也仅限于人类胰岛中的一部分 β 细胞，该子集在整个成年生命中都得以维持。 NNAT ^{+ β 细胞在成年阶段也显示出离散的转录组，代表专门用于胰岛素产生的亚群，并且在}db/db小鼠中减少。 NNAT ⁺群体中的“中心”细胞数量较少，这与这种功能特化的表观遗传控制一致。

结论/解释

这些发现表明， Nnat的差异 DNA 甲基化代表了一种在发育过程中建立 β 细胞异质性的新方法。因此，我们假设该位点甲基化的变化可能导致β细胞层次结构和连接性的丧失，从而可能导致某些形式的糖尿病中胰岛素分泌缺陷。

数据可用性

质谱蛋白质组学数据已通过 PRIDE 合作伙伴存储库存入 ProteomeXchange 联盟，数据集标识符为 PXD048465。

图形概要