Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC, Canada.
CELL Graduate Program, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
Diabetologia. 2022 Sep;65(9):1519-1533. doi: 10.1007/s00125-022-05718-1. Epub 2022 May 26.
AIMS/HYPOTHESIS: Pancreatic islets depend on cytosolic calcium (Ca) to trigger the secretion of glucoregulatory hormones and trigger transcriptional regulation of genes important for islet response to stimuli. To date, there has not been an attempt to profile Ca-regulated gene expression in all islet cell types. Our aim was to construct a large single-cell transcriptomic dataset from human islets exposed to conditions that would acutely induce or inhibit intracellular Ca signalling, while preserving biological heterogeneity.
We exposed intact human islets from three donors to the following conditions: (1) 2.8 mmol/l glucose; (2) 16 mmol/l glucose and 40 mmol/l KCl to maximally stimulate Ca signalling; and (3) 16 mmol/l glucose, 40 mmol/l KCl and 5 mmol/l EGTA (Ca chelator) to inhibit Ca signalling, for 1 h. We sequenced 68,650 cells from all islet cell types, and further subsetted the cells to form an endocrine cell-specific dataset of 59,373 cells expressing INS, GCG, SST or PPY. We compared transcriptomes across conditions to determine the differentially expressed Ca-regulated genes in each endocrine cell type, and in each endocrine cell subcluster of alpha and beta cells.
Based on the number of Ca-regulated genes, we found that each alpha and beta cell cluster had a different magnitude of Ca response. We also showed that polyhormonal clusters expressing both INS and GCG, or both INS and SST, are defined by Ca-regulated genes specific to each cluster. Finally, we identified the gene PCDH7 from the beta cell clusters that had the highest number of Ca-regulated genes, and showed that cells expressing cell surface PCDH7 protein have enhanced glucose-stimulated insulin secretory function.
CONCLUSIONS/INTERPRETATION: Here we use our large-scale, multi-condition, single-cell dataset to show that human islets have cell-type-specific Ca-regulated gene expression profiles, some of them specific to subpopulations. In our dataset, we identify PCDH7 as a novel marker of beta cells having an increased number of Ca-regulated genes and enhanced insulin secretory function.
A searchable and user-friendly format of the data in this study, specifically designed for rapid mining of single-cell RNA sequencing data, is available at https://lynnlab.shinyapps.io/Human_Islet_Atlas/ . The raw data files are available at NCBI Gene Expression Omnibus (GSE196715).
目的/假设：胰岛依赖细胞质钙（Ca）来触发葡萄糖调节激素的分泌，并触发对刺激的胰岛反应的基因的转录调节。迄今为止，还没有人试图对所有胰岛细胞类型的 Ca 调节基因表达进行分析。我们的目的是构建一个来自暴露于急性诱导或抑制细胞内 Ca 信号的条件的人类胰岛的大型单细胞转录组数据集，同时保留生物异质性。
我们将来自三个供体的完整人类胰岛暴露于以下条件：（1）2.8mmol/l 葡萄糖；（2）16mmol/l 葡萄糖和 40mmol/l KCl 以最大程度地刺激 Ca 信号；和（3）16mmol/l 葡萄糖、40mmol/l KCl 和 5mmol/l EGTA（Ca 螯合剂）抑制 Ca 信号，持续 1 小时。我们从所有胰岛细胞类型中测序了 68650 个细胞，并进一步细分细胞以形成一个包含 59373 个表达 INS、GCG、SST 或 PPY 的内分泌细胞特异性数据集。我们比较了不同条件下的转录组，以确定每个内分泌细胞类型以及 alpha 和 beta 细胞的每个内分泌细胞亚群中差异表达的 Ca 调节基因。
基于 Ca 调节基因的数量，我们发现每个 alpha 和 beta 细胞簇的 Ca 反应幅度不同。我们还表明，表达 INS 和 GCG 或 INS 和 SST 的多激素簇由每个簇特有的 Ca 调节基因定义。最后，我们从 beta 细胞簇中鉴定出具有最多 Ca 调节基因的基因 PCDH7，并表明表达细胞表面 PCDH7 蛋白的细胞具有增强的葡萄糖刺激胰岛素分泌功能。
结论/解释：在这里，我们使用我们的大规模、多条件、单细胞数据集表明，人类胰岛具有细胞类型特异性的 Ca 调节基因表达谱，其中一些是特定于亚群的。在我们的数据集，我们确定 PCDH7 作为一种新的标记物，具有更多的 Ca 调节基因和增强的胰岛素分泌功能的 beta 细胞。
本研究中数据的可搜索和用户友好格式，专门用于快速挖掘单细胞 RNA 测序数据，可在 https://lynnlab.shinyapps.io/Human_Islet_Atlas/ 上获得。原始数据文件可在 NCBI 基因表达综合数据库（GSE196715）中获得。
