Ins2 gene bursting activity defines a mature beta-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 beta-cells of the endocrine pancreas, including so-called extreme beta-cells exhibiting more than 2 fold higher insulin gene activity. However, it is not yet clear whether beta-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 beta-cells from Ins2GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, around 25 percent of beta-cells exhibited significantly higher activity at the conserved insulin gene Ins2(GFP)HIGH. Live-cell imaging captured on and off bursting behaviour in single beta-cells that lasted hours to days. Single cell RNA sequencing determined that Ins2(GFP)HIGH beta-cells were enriched for markers of beta-cell maturity and had reduced expression of anti-oxidant genes. Ins2(GFP)HIGH beta-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 beta-cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of beta-cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.