Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.
Publication Year:
2024
PubMed ID:
38512414
Public Summary:
This work was spear-headed by Dr. Steven J Millership and team at the Imperial College, London in which I had the opportunity to collaborate. For a very long time in the field, insulin-producing beta-cells in the pancreas were considered a very uniform population. However recent studies have established that there exists differences or heterogeneity within the beta-cell population in many aspects such as insulin secretion, cell-cell communication, self-renewal capacity. Interestingly, the proportion and distribution of these sub-populations are altered under diabetes. But a major knowledge gap in the field has been a lack of understanding of how this heterogeneity is established in the first place. This study established that a critical gene required for the production and release of insulin from beta-cells called Neuronatin (Nnat) is one of those factors that exhibits heterogeneous distribution in the beta-cells.
The team found that almost all beta-cells have very high levels of Nnat in the fetal stages. Contrarily, most beta-cells in the adults have very low Nnat levels, except for a small subset that is able to retain high Nnat levels (NnatHigh). Interestingly, this NnatHigh beta-cells had distinct characteristics as compared to the more populous NnatLow beta-cells and eliminating them severely affected the ability to efficiently release insulin in response to rising glucose levels. The study also identified a novel mechanism by which this unique population is established and maintained, namely the changes in the “epigenome” by methylation of genomic DNA, an aspect that our lab specializes in. My specific contribution to this study was to determine if the proportion of beta-cell population with high and low levels of Nnat levels is altered under diabetes. Using a mouse model for diabetes and respective controls, I found that the distinct high-low Nnat pattern exhibited by healthy adult beta-cell population was lost under diabetes. Therefore this study established that differences in methylation pattern on DNA is a key mechanism that results in beta-cell heterogeneity.
Scientific Abstract:
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 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.
