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 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 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.

Delineating mouse β-cell identity during lifetime and in diabetes with a single cell atlas

“…Group 1 contained genes implicated in β-cell metabolic stress recovery, such as ATP production related genes 101 ( Figure 5g , Supplementary Table S7 ). The negative correlation between expression of group 1 and groups 3/5 is in accordance with previously reported cycling of β-cells between insulin production and recovery in mouse and human 101, 106, 107 . As group 1 genes, including multiple mitochondrial genes, β-cell maturation and function genes ( Ucn3 , Ftl1 , Cd63 , Scg2) 73, 108 , and protective genes ( Nupr1 , Atp2a2 , Atf5 ) 109–111 , are involved in healthy metabolic stress recovery they may be of interest for T2D therapy.…”

Signal transduction pathways controllingIns2gene activity and β cell state transitions

“…2B). These results suggest that cells in the less mature Ins2 (GFP) LOW state was enriched in markers of ‘multi-hormonal’ cells, consistent with our previous scRNAseq analysis (15). We also found enrichment in proteins in endoplasmic reticulum processing, including HSPA5, HSP90B1, and ERO1L.…”

Sex differences in islet stress responses support female β cell resilience

“…Because insulin biosynthesis accounts for approximately half the total protein production in β cells [ 88 ], one potential explanation for the sex-specific recovery from protein synthesis repression is a sex difference in transcriptional changes to insulin. To test this, we quantified GFP levels in β cells isolated from male and female mice with GFP knocked into the endogenous mouse Ins2 locus ( Ins2 GFP/WT ) [ 51 , 89 ]. While ER stress induced a significant reduction in Ins2 gene activity, this response was equivalent between the sexes.…”

“…To measure cell death, islet cells were imaged every 2 h for 84 h. MetaXpress software was used to quantify cell death, defined as the number of Propidium Iodide-positive/Hoechst 33342-positive cells. To measure Ins2 gene activity, dispersed islets from Ins2 GFP/WT mice aged 21–23 weeks were used [ 51 ]. Islet cells were imaged every 30 min for 60 h. MetaXpress analysis software and custom R scripts were used to perform single-cell tracking of Ins2 GFP/WT β cells as previously described [ 51 ].…”

“…To measure Ins2 gene activity, dispersed islets from Ins2 GFP/WT mice aged 21–23 weeks were used [ 51 ]. Islet cells were imaged every 30 min for 60 h. MetaXpress analysis software and custom R scripts were used to perform single-cell tracking of Ins2 GFP/WT β cells as previously described [ 51 ].…”