Ins2 gene bursting activity defines a mature β-cell state

Heterogeneity within specific cell types is common and increasingly apparent with the advent of single-cell transcriptomics. Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas, including so-called extreme β-cells exhibiting >2 fold higher insulin gene activity. However, it is not yet clear whether β-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live-cell imaging, with complementary experiments employing FACS and single-cell RNA sequencing, in β-cells from Ins2 GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2 GFP mice confirmed that at a given moment, ∼25% of β-cells exhibited significantly higher activity at the conserved insulin gene Ins2 (GFP) HIGH . Live-cell imaging captured on and off ‘bursting’ behaviour in single β-cells that lasted hours to days. Single cell RNA sequencing determined that Ins2 (GFP) HIGH β-cells were enriched for markers of β-cell maturity and had reduced expression of anti-oxidant genes. Ins2 (GFP) HIGH β-cells were also significantly less viable at all glucose concentrations and in the context of ER stress. Collectively, our results demonstrate that the heterogeneity of extreme insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of β-cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.