Dynamic Ins2 gene activity defines β-cell maturity states

Transcriptional and functional cellular specialization has been described for insulin-secreting β-cells of the endocrine pancreas. However, it is not 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 Ins2GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ~25% of β-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single β-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 hours. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single cell RNA sequencing showed that Ins2(GFP)HIGH β-cells were enriched for markers of β-cell maturity. 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 insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity.