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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.


Single cell based calcium influx analysis reveals a quantitative difference in glucose responsive cells between WT and diabetic mouse islets.(A-C) Representative images showing the number of single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in (A) WT mouse islets, (B) db/db mouse islets, and (C) NOD mouse islets. (D-F) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 islets: (D) WT mouse islet cells (total number of cells analyzed from each islet was n = 216, n = 190, n = 144), (E) db/db mouse islet cells (n = 239, n = 132, n = 113), and (F) NOD mouse islet cells (n = 69, n = 64, n = 50). The WT islets had on average 53±9% of fully responsive cells and 4±3% of non-responsive cells, while db/db and NOD islets on average had 1±1% and 9±3% fully responsive cells and 59±10% and 23±6% non-responsive cells accordingly. Scale bar = 100 μm.
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pone.0122044.g003: Single cell based calcium influx analysis reveals a quantitative difference in glucose responsive cells between WT and diabetic mouse islets.(A-C) Representative images showing the number of single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in (A) WT mouse islets, (B) db/db mouse islets, and (C) NOD mouse islets. (D-F) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 islets: (D) WT mouse islet cells (total number of cells analyzed from each islet was n = 216, n = 190, n = 144), (E) db/db mouse islet cells (n = 239, n = 132, n = 113), and (F) NOD mouse islet cells (n = 69, n = 64, n = 50). The WT islets had on average 53±9% of fully responsive cells and 4±3% of non-responsive cells, while db/db and NOD islets on average had 1±1% and 9±3% fully responsive cells and 59±10% and 23±6% non-responsive cells accordingly. Scale bar = 100 μm.

Mentions: Calcium responsiveness of individual cells was assessed based on the number of responses to three glucose challenges (Fig 3). Cells that flux calcium in response to all three glucose stimulations are circled in red, cells that responded once or twice are circled orange, and cells that did not respond are circled green (Fig 3A–3C). For WT mouse islets, there was a large proportion of more responsive red and orange cells positioned close to each other and the few non-responsive green cells were typically found at the islet’s edge (Fig 3A). In comparison, db/db and NOD islets had random positioning of red, orange, and green cells (Fig 3B and 3C). WT islets predominantly contained cells that responded to all three glucose stimulations (Fig 3D), whereas cells from diabetic islets responded to glucose less frequently or not at all (Fig 3E and 3F).


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

Kenty JH, Melton DA - PLoS ONE (2015)

Single cell based calcium influx analysis reveals a quantitative difference in glucose responsive cells between WT and diabetic mouse islets.(A-C) Representative images showing the number of single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in (A) WT mouse islets, (B) db/db mouse islets, and (C) NOD mouse islets. (D-F) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 islets: (D) WT mouse islet cells (total number of cells analyzed from each islet was n = 216, n = 190, n = 144), (E) db/db mouse islet cells (n = 239, n = 132, n = 113), and (F) NOD mouse islet cells (n = 69, n = 64, n = 50). The WT islets had on average 53±9% of fully responsive cells and 4±3% of non-responsive cells, while db/db and NOD islets on average had 1±1% and 9±3% fully responsive cells and 59±10% and 23±6% non-responsive cells accordingly. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390334&req=5

pone.0122044.g003: Single cell based calcium influx analysis reveals a quantitative difference in glucose responsive cells between WT and diabetic mouse islets.(A-C) Representative images showing the number of single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in (A) WT mouse islets, (B) db/db mouse islets, and (C) NOD mouse islets. (D-F) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 islets: (D) WT mouse islet cells (total number of cells analyzed from each islet was n = 216, n = 190, n = 144), (E) db/db mouse islet cells (n = 239, n = 132, n = 113), and (F) NOD mouse islet cells (n = 69, n = 64, n = 50). The WT islets had on average 53±9% of fully responsive cells and 4±3% of non-responsive cells, while db/db and NOD islets on average had 1±1% and 9±3% fully responsive cells and 59±10% and 23±6% non-responsive cells accordingly. Scale bar = 100 μm.
Mentions: Calcium responsiveness of individual cells was assessed based on the number of responses to three glucose challenges (Fig 3). Cells that flux calcium in response to all three glucose stimulations are circled in red, cells that responded once or twice are circled orange, and cells that did not respond are circled green (Fig 3A–3C). For WT mouse islets, there was a large proportion of more responsive red and orange cells positioned close to each other and the few non-responsive green cells were typically found at the islet’s edge (Fig 3A). In comparison, db/db and NOD islets had random positioning of red, orange, and green cells (Fig 3B and 3C). WT islets predominantly contained cells that responded to all three glucose stimulations (Fig 3D), whereas cells from diabetic islets responded to glucose less frequently or not at all (Fig 3E and 3F).

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.