Dynamic Ins2 Gene Activity Defines beta-Cell Maturity States

Transcriptional and functional cellular specialization has been described for insulin-secreting beta-cells of the endocrine pancreas. However, it is not 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 using FACS and single-cell RNA sequencing, in beta-cells from Ins2(GFP) knockin mice. In vivo staining and FACS analysis of islets from Ins2(GFP) mice confirmed that at a given moment, similar to 25% of beta-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single b-cells. Autocorrelation analysis revealed a subset of oscillating cells, withmean oscillation periods of 17 h. 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) beta-cells were enriched for markers of b-cell maturity. Ins2(GFP)(HIGH) beta-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed inmouse and human beta-cells, can be accounted for by dynamic states of insulin gene activity.