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Additional file 1: Table S1. Primary and secondary antibodies.

Additional file 4: Fig. S2. scRNAseq analysis shows there are multiple cell types in the proliferating cell cluster and there are 3 types of fibroblasts. A Unsupervised sub-clustering of the cluster containing proliferating cells, represented as an UMAP plot. The proliferating cell cluster contains proliferating T cells, B cells, Na��ve B cells, NK cells and epithelial cells. B Numbers of cells from PK-Ins1-/-;Ins2+/+ (orange) and PK-Ins1-/-;Ins2+/- (green) mice for each cell type. C Violin plot showing the expression level of selected cell type-specific markers for identified cell types within the proliferating cell cluster. D Expression level of the typical markers for identifying M1 macrophages and M2 macrophages for each genotype. E Violin plots showing the expression level of selected markers for inflammatory, myofibroblastic and antigen-presenting fibroblasts. F The differential expression of Reg3a, Reg3b, Reg3d, and Reg3g genes in acinar cells between PK-Ins1-/-;Ins2+/+ and PK-Ins1-/-;Ins2+/- mice.

Additional file 3: Fig. S1. Amylase activity and pancreatic area of PK-Ins1-/-;Ins2+/+ and PK-Ins1-/-;Ins2+/- mice and the sex difference in PanIN development. A-B The amylase activity in male (A) and female mice (B) for each genotype. C-D The comparison of percent of total pancreatic area occupied by PanINs and tumor (C) or only PanINs (excluding tumor bearing mice) (D) in male and female PK-Ins1-/-; Ins2+/+ mice (n= 15-22) (dark blue and dark orange dots denote mice that developed tumors). E The comparison of percent of total pancreatic area occupied by only PanINs in male and female PK-Ins1-/-; Ins2+/- mice (n= 10-14). F The comparison of percent of total pancreatic area occupied by ADM in male and female PK-Ins1-/-; Ins2+/+ mice (n= 16-22). G The comparison of percent of total pancreatic area occupied by ADM in male and female PK-Ins1-/-; Ins2+/- mice (n= 10-14). H The total pancreatic area for mice in an Ins2-null background or in an Ins1-null background. I-J Correlations of adipocyte area with PanIN area in non-tumor bearing male (I) and female (J) PK-Ins1-/-;Ins2+/+ (dark colors) and PK-Ins1-/-;Ins2+/- (light colors) mice (n = 10-22). ****p

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 β-cells of the endocrine pancreas, including so-called extreme β-cells exhibiting >2 fold higher insulin gene activity. However, it is not yet 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 (GFP) HIGH . Live-cell imaging captured on and off ‘bursting’ behaviour in single β-cells that lasted hours to days. Single cell RNA sequencing determined that Ins2 (GFP) HIGH β-cells were enriched for markers of β-cell maturity and had reduced expression of anti-oxidant genes. 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 extreme insulin production, observed in mouse and human β-cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of β-cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.