Dynamic Ins2 Gene Activity Defines β-Cell Maturity States

Abstract

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 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. Live cell imaging captured Ins2 gene activity dynamics in single β-cells over time. Autocorrelation analysis revealing a subset of cells with oscillating behavior, with 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 determined 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.