Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

• Published in: Diabetologia 2024-06, Vol.67 (6), p.1079-1094
• Main Authors: Yu, Vanessa, Yong, Fiona, Marta, Angellica, Khadayate, Sanjay, Osakwe, Adrien, Bhattacharya, Supriyo, Varghese, Sneha S., Chabosseau, Pauline, Tabibi, Sayed M., Chen, Keran, Georgiadou, Eleni, Parveen, Nazia, Suleiman, Mara, Stamoulis, Zoe, Marselli, Lorella, De Luca, Carmela, Tesi, Marta, Ostinelli, Giada, Delgadillo-Silva, Luis, Wu, Xiwei, Hatanaka, Yuki, Montoya, Alex, Elliott, James, Patel, Bhavik, Demchenko, Nikita, Whilding, Chad, Hajkova, Petra, Shliaha, Pavel, Kramer, Holger, Ali, Yusuf, Marchetti, Piero, Sladek, Robert, Dhawan, Sangeeta, Withers, Dominic J., Rutter, Guy A., Millership, Steven J.
• Format: Article
• Language: English
• Subjects: Animals; Beta cells; Calcium signalling; CpG islands; CpG Islands - genetics; Developmental stages; Diabetes; Diabetes mellitus (non-insulin dependent); DNA Methylation; DNA methyltransferase; DNA Methyltransferase 3A - metabolism; Embryogenesis; Epigenetics; Flow cytometry; Human Physiology; Humans; Insulin; Insulin - metabolism; Insulin secretion; Insulin Secretion - physiology; Insulin-Secreting Cells - metabolism; Internal Medicine; Mass spectrometry; Mass spectroscopy; Medicine; Medicine & Public Health; Membrane Proteins - genetics; Membrane Proteins - metabolism; Metabolic Diseases; Mice; Mice, Transgenic; Molecular modelling; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neural networks; Pancreas; Proteomics; Scientific imaging; Secretion; Transcriptomes; Transgenic mice
• ISSN: 0012-186X; 1432-0428; 1432-0428
• DOI: 10.1007/s00125-024-06123-6

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 Ca 2+ 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 Ca 2+ 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 hypo