Ins2 gene bursting activity defines a mature β-cell state

AbstractHeterogeneity 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 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(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.

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SCOPE-Seq: a scalable technology for linking live cell imaging and single-cell RNA sequencing

Genome Biology ◽

10.1186/s13059-018-1607-x ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 15

Author(s):

Jinzhou Yuan ◽

Jenny Sheng ◽

Peter A. Sims

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Single Cell Rna Sequencing

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Single-cell RNA sequencing reveals a role for reactive oxygen species and peroxiredoxins in fatty-acid-induced rat β-cell proliferation

10.1101/2021.12.22.473904 ◽

2021 ◽

Author(s):

Alexis Vivoli ◽

Julien Ghislain ◽

Ali Filali-Mouhim ◽

Zuraya Elisa Angeles ◽

Anne-Laure Castell ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Proliferation ◽

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Reactive Oxygen ◽

Oxygen Species ◽

Β Cells ◽

Β Cell ◽

Single Cell Rna Sequencing

The functional mass of insulin-secreting pancreatic β cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing β cells. To decipher the underlying molecular mechanisms, we assessed β-cell proliferation in isolated rat islets exposed to glucose and oleate or palmitate for 48 h and analyzed the transcriptional response by single-cell RNA sequencing. Unsupervised clustering of pooled βcells identified subpopulations, including proliferating β cells. β-cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved energy metabolism and mitochondrial activity. Comparison of proliferating vs. non-proliferating β cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, the antioxidant N-acetyl cysteine and the peroxiredoxin inhibitor Conoidin A both blocked oleate-induced β-cell proliferation. Our data reveal a key role for ROS signaling in β-cell proliferation in response to nutrients.

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Endocrine Pancreas Development and Dysfunction Through the Lens of Single-Cell RNA-Sequencing

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.629212 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wojciech J. Szlachcic ◽

Natalia Ziojla ◽

Dorota K. Kizewska ◽

Marcelina Kempa ◽

Malgorzata Borowiak

Keyword(s):

Blood Glucose ◽

Single Cell ◽

Rna Sequencing ◽

Endocrine Pancreas ◽

Pancreas Development ◽

Β Cells ◽

Β Cell ◽

Regulatory Pathways ◽

Endocrine Differentiation ◽

Single Cell Rna Sequencing

A chronic inability to maintain blood glucose homeostasis leads to diabetes, which can damage multiple organs. The pancreatic islets regulate blood glucose levels through the coordinated action of islet cell-secreted hormones, with the insulin released by β-cells playing a crucial role in this process. Diabetes is caused by insufficient insulin secretion due to β-cell loss, or a pancreatic dysfunction. The restoration of a functional β-cell mass might, therefore, offer a cure. To this end, major efforts are underway to generate human β-cells de novo, in vitro, or in vivo. The efficient generation of functional β-cells requires a comprehensive knowledge of pancreas development, including the mechanisms driving cell fate decisions or endocrine cell maturation. Rapid progress in single-cell RNA sequencing (scRNA-Seq) technologies has brought a new dimension to pancreas development research. These methods can capture the transcriptomes of thousands of individual cells, including rare cell types, subtypes, and transient states. With such massive datasets, it is possible to infer the developmental trajectories of cell transitions and gene regulatory pathways. Here, we summarize recent advances in our understanding of endocrine pancreas development and function from scRNA-Seq studies on developing and adult pancreas and human endocrine differentiation models. We also discuss recent scRNA-Seq findings for the pathological pancreas in diabetes, and their implications for better treatment.

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Single-cell RNA sequencing of mouse islets exposed to proinflammatory cytokines

Life Science Alliance ◽

10.26508/lsa.202000949 ◽

2021 ◽

Vol 4 (6) ◽

pp. e202000949

Author(s):

Jennifer S Stancill ◽

Moujtaba Y Kasmani ◽

Achia Khatun ◽

Weiguo Cui ◽

John A Corbett

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rna Sequencing ◽

Proinflammatory Cytokines ◽

Cell Damage ◽

Cytokine Signaling ◽

Β Cells ◽

Antiviral Genes ◽

Single Cell Rna Sequencing ◽

Mouse Islets

Exposure to proinflammatory cytokines is believed to contribute to pancreatic β-cell damage during diabetes development. Although some cytokine-mediated changes in islet gene expression are known, the heterogeneity of the response is not well-understood. After 6-h treatment with IL-1β and IFN-γ alone or together, mouse islets were subjected to single-cell RNA sequencing. Treatment with both cytokines together led to expression of inducible nitric oxide synthase mRNA (Nos2) and antiviral and immune-associated genes in a subset of β-cells. Interestingly, IL-1β alone activated antiviral genes. Subsets of δ- and α-cells expressed Nos2 and exhibited similar gene expression changes as β-cells, including increased expression of antiviral genes and repression of identity genes. Finally, cytokine responsiveness was inversely correlated with expression of genes encoding heat shock proteins. Our findings show that all islet endocrine cell types respond to cytokines, IL-1β induces the expression of protective genes, and cellular stress gene expression is associated with inhibition of cytokine signaling.

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