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Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression.

Kenty JH, Melton DA - PLoS ONE (2015)

Bottom Line: Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression.Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells.Human islet cells were also assessed and showed functional α and β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.

No MeSH data available.


Related in: MedlinePlus

Population and single cell-based calcium influx analysis show defects in diabetic mouse islets.(A-C) Representative images of analysis selection setting for population (left) and single cell (right) analysis for (A) WT mouse islet, (B) db/db mouse islet, and (C) NOD mouse islet. Scale bar = 100 μm. Note: Fed blood glucose level of the db/db and NOD mice was > 550 mg/dL. (D-F) Population measurements of dynamic normalized Fluo-4 fluorescence intensity for one islet (out of three islets analyzed for each mouse strain): (D) WT mouse islet, (E) db/db mouse islet, and (F) NOD mouse islet calcium imaging during sequential glucose stimulation. (G-I) Single cell measurements of dynamic Fluo-4 fluorescence intensity for (G) WT mouse islets, (H) db/db mouse islets, and (I) NOD mouse islets upon calcium imaging during glucose challenges.
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pone.0122044.g002: Population and single cell-based calcium influx analysis show defects in diabetic mouse islets.(A-C) Representative images of analysis selection setting for population (left) and single cell (right) analysis for (A) WT mouse islet, (B) db/db mouse islet, and (C) NOD mouse islet. Scale bar = 100 μm. Note: Fed blood glucose level of the db/db and NOD mice was > 550 mg/dL. (D-F) Population measurements of dynamic normalized Fluo-4 fluorescence intensity for one islet (out of three islets analyzed for each mouse strain): (D) WT mouse islet, (E) db/db mouse islet, and (F) NOD mouse islet calcium imaging during sequential glucose stimulation. (G-I) Single cell measurements of dynamic Fluo-4 fluorescence intensity for (G) WT mouse islets, (H) db/db mouse islets, and (I) NOD mouse islets upon calcium imaging during glucose challenges.

Mentions: Calcium imaging was utilized to compare glucose-stimulated calcium influx in the islets of WT and diabetic mice at the population and single cell levels (Fig 2). WT islets were obtained from normal ICR mice, Type II diabetic islets were obtained from db/db mice, and Type I diabetic islets were obtained from NOD mice. Fig 2A–2C show the population and single cell analysis of calcium imaging. WT islets fluxed calcium in response to high glucose at the population level (Fig 2D) and with well-synchronized responses among single cells (Fig 2G and S3 Movie). In contrast, db/db and NOD islets showed weak calcium responses to glucose stimulation at the population level (Fig 2E, 2F and S4 Movie) and lack the synchronous phenotype observed at the single cell level (Fig 2H, 2I and S5 Movie). A comparison of Fig 2D–2G to Fig 2E–2I shows a clear defect in the diabetic islet cells with respect to dynamic and coordinated calcium flux.


Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression.

Kenty JH, Melton DA - PLoS ONE (2015)

Population and single cell-based calcium influx analysis show defects in diabetic mouse islets.(A-C) Representative images of analysis selection setting for population (left) and single cell (right) analysis for (A) WT mouse islet, (B) db/db mouse islet, and (C) NOD mouse islet. Scale bar = 100 μm. Note: Fed blood glucose level of the db/db and NOD mice was > 550 mg/dL. (D-F) Population measurements of dynamic normalized Fluo-4 fluorescence intensity for one islet (out of three islets analyzed for each mouse strain): (D) WT mouse islet, (E) db/db mouse islet, and (F) NOD mouse islet calcium imaging during sequential glucose stimulation. (G-I) Single cell measurements of dynamic Fluo-4 fluorescence intensity for (G) WT mouse islets, (H) db/db mouse islets, and (I) NOD mouse islets upon calcium imaging during glucose challenges.
© Copyright Policy
Related In: Results  -  Collection

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pone.0122044.g002: Population and single cell-based calcium influx analysis show defects in diabetic mouse islets.(A-C) Representative images of analysis selection setting for population (left) and single cell (right) analysis for (A) WT mouse islet, (B) db/db mouse islet, and (C) NOD mouse islet. Scale bar = 100 μm. Note: Fed blood glucose level of the db/db and NOD mice was > 550 mg/dL. (D-F) Population measurements of dynamic normalized Fluo-4 fluorescence intensity for one islet (out of three islets analyzed for each mouse strain): (D) WT mouse islet, (E) db/db mouse islet, and (F) NOD mouse islet calcium imaging during sequential glucose stimulation. (G-I) Single cell measurements of dynamic Fluo-4 fluorescence intensity for (G) WT mouse islets, (H) db/db mouse islets, and (I) NOD mouse islets upon calcium imaging during glucose challenges.
Mentions: Calcium imaging was utilized to compare glucose-stimulated calcium influx in the islets of WT and diabetic mice at the population and single cell levels (Fig 2). WT islets were obtained from normal ICR mice, Type II diabetic islets were obtained from db/db mice, and Type I diabetic islets were obtained from NOD mice. Fig 2A–2C show the population and single cell analysis of calcium imaging. WT islets fluxed calcium in response to high glucose at the population level (Fig 2D) and with well-synchronized responses among single cells (Fig 2G and S3 Movie). In contrast, db/db and NOD islets showed weak calcium responses to glucose stimulation at the population level (Fig 2E, 2F and S4 Movie) and lack the synchronous phenotype observed at the single cell level (Fig 2H, 2I and S5 Movie). A comparison of Fig 2D–2G to Fig 2E–2I shows a clear defect in the diabetic islet cells with respect to dynamic and coordinated calcium flux.

Bottom Line: Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression.Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells.Human islet cells were also assessed and showed functional α and β cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Harvard Stem Cell Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.

No MeSH data available.


Related in: MedlinePlus