Glucokinase activity controls subpopulations of β-cells that alternately lead islet Ca2+ oscillations
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eLife Assessment
This study provides convincing evidence for functional subpopulations of β-cells responsible for Ca2+ signal initiation and maintenance using novel three-dimensional light sheet microscopy imaging and analysis of pancreatic islets. The findings are important as they help decode the mechanistic underpinnings of islet calcium oscillations and the resulting pulsatile insulin secretion. The work will be of general interest to cell biologists and of particular interest to islet biologists.
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Abstract
Oscillations in insulin secretion, driven by islet Ca 2+ waves, are crucial for glycemic control. Prior studies, performed with single-plane imaging, suggest that subpopulations of electrically coupled β-cells have privileged roles in leading and coordinating the propagation of Ca 2+ waves. Here, we used 3D light-sheet imaging to analyze the location and Ca 2+ activity of single β-cells within the entire islet at >2 Hz. In contrast with single-plane studies, 3D network analysis indicates that the most highly synchronized β-cells are located at the islet center, and remain regionally but not cellularly stable between oscillations. This subpopulation, which includes ‘hub cells’, is insensitive to changes in fuel metabolism induced by glucokinase and pyruvate kinase activation. β-cells that initiate the Ca 2+ wave (‘leaders’) are located at the islet periphery, and strikingly, change their identity over time via rotations in the wave axis. Glucokinase activation, which increased oscillation period, reinforced leader cells and stabilized the wave axis. Pyruvate kinase activation, despite increasing oscillation frequency, had no effect on leader cells, indicating the wave origin is patterned by fuel input. These findings emphasize the stochastic nature of the β-cell subpopulations that control Ca 2+ oscillations and identify a role for glucokinase in spatially patterning ‘leader’ β-cells.
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eLife Assessment
This study provides convincing evidence for functional subpopulations of β-cells responsible for Ca2+ signal initiation and maintenance using novel three-dimensional light sheet microscopy imaging and analysis of pancreatic islets. The findings are important as they help decode the mechanistic underpinnings of islet calcium oscillations and the resulting pulsatile insulin secretion. The work will be of general interest to cell biologists and of particular interest to islet biologists.
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Reviewer #1 - Public Review
Summary:
Jin, Briggs, and colleagues use light sheet imaging to reconstruct the islet three-dimensional Ca2+ network. The authors find that early/late responding (leader) cells are dynamic over time, and located at the islet periphery. By contrast, highly connected or hub cells are stable and located toward the islet center. Suggesting that the two subpopulations are differentially regulated by fuel input, glucokinase activation only influences leader cell phenotype, whereas hubs remain stable.
Strengths:
The studies are novel in providing the first three-dimensional snapshot of the beta cell functional network, as well as determining the localization of some of the different subpopulations identified to date. The studies also provide some consensus as to the origin, stability, and role of such subpopulations …
Reviewer #1 - Public Review
Summary:
Jin, Briggs, and colleagues use light sheet imaging to reconstruct the islet three-dimensional Ca2+ network. The authors find that early/late responding (leader) cells are dynamic over time, and located at the islet periphery. By contrast, highly connected or hub cells are stable and located toward the islet center. Suggesting that the two subpopulations are differentially regulated by fuel input, glucokinase activation only influences leader cell phenotype, whereas hubs remain stable.
Strengths:
The studies are novel in providing the first three-dimensional snapshot of the beta cell functional network, as well as determining the localization of some of the different subpopulations identified to date. The studies also provide some consensus as to the origin, stability, and role of such subpopulations in islet function.
Weaknesses:
Experiments with metabolic enzyme activators do not take into account the influence of cell viability on the observed Ca2+ network data. Limitations of the imaging approach used need to be recognized and evaluated/discussed.
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Reviewer #2 - Public Review
The manuscript by Erli Jin, Jennifer Briggs et al. utilizes light sheet microscopy to image islet beta cell calcium oscillations in 3D and determine where beta cell populations are located that begin and coordinate glucose-stimulated calcium oscillations. The light sheet technique allowed clear 3D mapping of beta cell calcium responses to glucose, glucokinase activation, and pyruvate kinase activation. The manuscript finds that synchronized beta-cells are found at the islet center, that leader beta cells showing the first calcium responses are located on the islet periphery, that glucokinase activation helped maintain beta cells that lead calcium responses, and that pyruvate kinase activation primarily increases islet calcium oscillation frequency. The study is well-designed, contains a significant amount of …
Reviewer #2 - Public Review
The manuscript by Erli Jin, Jennifer Briggs et al. utilizes light sheet microscopy to image islet beta cell calcium oscillations in 3D and determine where beta cell populations are located that begin and coordinate glucose-stimulated calcium oscillations. The light sheet technique allowed clear 3D mapping of beta cell calcium responses to glucose, glucokinase activation, and pyruvate kinase activation. The manuscript finds that synchronized beta-cells are found at the islet center, that leader beta cells showing the first calcium responses are located on the islet periphery, that glucokinase activation helped maintain beta cells that lead calcium responses, and that pyruvate kinase activation primarily increases islet calcium oscillation frequency. The study is well-designed, contains a significant amount of high-quality data, and the conclusions are largely supported by the results.
It has recently been shown that beta cells within islets containing intact vasculature (such as those in a pancreatic slice) show different calcium responses compared to isolated islets (such as that shown in PMID: 35559734). It would be important to include some discussion about the potential in vitro artifacts in calcium that arise following islet isolation (this could be included in the discussion about the limitations of the study).
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Reviewer #3 - Public Review
Summary:
Jin, Briggs et al. made use of light-sheet 3D imaging and data analysis to assess the collective network activity in isolated mouse islets. The major advantage of using whole islet imaging, despite compromising on the speed of acquisition, is that it provides a complete description of the network, while 2D networks are only an approximation of the islet network. In static-incubation conditions, excluding the effects of perfusion, they assessed two subpopulations of beta cells and their spatial consistency and metabolic dependence.
Strengths:
The authors confirmed that coordinated Ca2+ oscillations are important for glycemic control. In addition, they definitively disproved the role of individual privileged cells, which were suggested to lead or coordinate Ca²⁺ oscillations. They provided evidence for …
Reviewer #3 - Public Review
Summary:
Jin, Briggs et al. made use of light-sheet 3D imaging and data analysis to assess the collective network activity in isolated mouse islets. The major advantage of using whole islet imaging, despite compromising on the speed of acquisition, is that it provides a complete description of the network, while 2D networks are only an approximation of the islet network. In static-incubation conditions, excluding the effects of perfusion, they assessed two subpopulations of beta cells and their spatial consistency and metabolic dependence.
Strengths:
The authors confirmed that coordinated Ca2+ oscillations are important for glycemic control. In addition, they definitively disproved the role of individual privileged cells, which were suggested to lead or coordinate Ca²⁺ oscillations. They provided evidence for differential regional stability, confirming the previously described stochastic nature of the beta cells that act as strongly connected hubs as well as beta cells in initiating regions (doi.org/10.1103/PhysRevLett.127.168101).
The fact that islet cores contain beta cells that are more active and more coordinated has also been readily observed in high-frequency 2D recordings (e.g. DOI: 10.2337/db22-0952), suggesting that the high-speed capture of fast activity can partially compensate for incomplete topological information.
They also found an increased metabolic sensitivity of mantle regions of an islet with a subpopulation of beta cells with a high probability of leading the islet activity which can be entrained by fuel input. They discuss a potential role of alpha/delta cell interaction, however relative lack of beta cells in the islet border region could also be a factor contributing to less connectivity and higher excitability.
The Methods section contains a useful series of direct instructions on how to approach fast 3D imaging with currently available hardware and software.
The Discussion is clear and includes most of the issues regarding the interpretation of the presented results.
Some issues concerning inconsistencies between data presented and statements made as well as statistical analysis need to be addressed.
Taken together it is a strong technical paper to demonstrate the stochasticity regarding the functions subpopulations of beta cells in the islets may have and how less well-resolved approaches (both missing spatial resolution as well as missing temporal resolution) led us to jump to unjustified conclusions regarding the fixed roles of individual beta cells within an islet.
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