Limits...
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.


Human islets contain cells that influx calcium in response to multiple glucose challenges and show expression of β cell markers.(A) Representative images of analysis selection setting for population (left) and single cell (right) analysis for a human islet. Scale bar = 100 μm. (B) Representative population measurements of dynamic normalized Fluo-4 fluorescence intensity for one human islet (out of three islets analyzed). (C) Single cell measurements of dynamic Fluo-4 fluorescence intensity for human islets (from the same donor). (D) Representative images showing single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in human islets. (E) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 human islets (total number of cells analyzed from each islet was n = 245, n = 201, and n = 176). On average, WT human islets had 51±4% that responded 3 times, 33±6% that responded 2 or 1 times, and 15±5% that responded to no glucose challenge. (F) Marker expression profiles and responsiveness to glucose for individual cells of human islets. Color scheme is the same as Fig 4.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390334&req=5

pone.0122044.g005: Human islets contain cells that influx calcium in response to multiple glucose challenges and show expression of β cell markers.(A) Representative images of analysis selection setting for population (left) and single cell (right) analysis for a human islet. Scale bar = 100 μm. (B) Representative population measurements of dynamic normalized Fluo-4 fluorescence intensity for one human islet (out of three islets analyzed). (C) Single cell measurements of dynamic Fluo-4 fluorescence intensity for human islets (from the same donor). (D) Representative images showing single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in human islets. (E) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 human islets (total number of cells analyzed from each islet was n = 245, n = 201, and n = 176). On average, WT human islets had 51±4% that responded 3 times, 33±6% that responded 2 or 1 times, and 15±5% that responded to no glucose challenge. (F) Marker expression profiles and responsiveness to glucose for individual cells of human islets. Color scheme is the same as Fig 4.

Mentions: Calcium imaging during multiple glucose challenges was performed on intact human islets which were subsequently stained for marker expression (Fig 5). Population and single cell calcium imaging of human islets show responses to high glucose but with less synchronized responses compared to that of WT mouse islets (Fig 5A and 5B, 5C and S9 Movie). Similar to WT mouse islets, human islets that responded to all 3 high glucose stimulations were mostly β cells, as determined by INS/NKX6.1 coexpression. Cells that did not respond to high glucose showed an inversed calcium response to low glucose and expressed α cell markers (Fig 5D, 5E, and 5F). Human islets also contained dysfunctional β cells that showed partial calcium responses to glucose challenges with lower calcium influx peaks compared to fully calcium responsive cells (Fig 5F). Unlike WT mouse islets, human islets contained cells expressing INS marker alone and these showed three significant responses to high glucose challenges, albeit with lower calcium peaks than the response of double positive, INS/NKX6.1 human β cells.


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

Kenty JH, Melton DA - PLoS ONE (2015)

Human islets contain cells that influx calcium in response to multiple glucose challenges and show expression of β cell markers.(A) Representative images of analysis selection setting for population (left) and single cell (right) analysis for a human islet. Scale bar = 100 μm. (B) Representative population measurements of dynamic normalized Fluo-4 fluorescence intensity for one human islet (out of three islets analyzed). (C) Single cell measurements of dynamic Fluo-4 fluorescence intensity for human islets (from the same donor). (D) Representative images showing single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in human islets. (E) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 human islets (total number of cells analyzed from each islet was n = 245, n = 201, and n = 176). On average, WT human islets had 51±4% that responded 3 times, 33±6% that responded 2 or 1 times, and 15±5% that responded to no glucose challenge. (F) Marker expression profiles and responsiveness to glucose for individual cells of human islets. Color scheme is the same as Fig 4.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122044.g005: Human islets contain cells that influx calcium in response to multiple glucose challenges and show expression of β cell markers.(A) Representative images of analysis selection setting for population (left) and single cell (right) analysis for a human islet. Scale bar = 100 μm. (B) Representative population measurements of dynamic normalized Fluo-4 fluorescence intensity for one human islet (out of three islets analyzed). (C) Single cell measurements of dynamic Fluo-4 fluorescence intensity for human islets (from the same donor). (D) Representative images showing single cells that responded to 3 (red), 2 or 1 (orange), and 0 (green) glucose challenges in human islets. (E) Quantification of the frequency of cells responding to 15 mM glucose analyzed from 3 human islets (total number of cells analyzed from each islet was n = 245, n = 201, and n = 176). On average, WT human islets had 51±4% that responded 3 times, 33±6% that responded 2 or 1 times, and 15±5% that responded to no glucose challenge. (F) Marker expression profiles and responsiveness to glucose for individual cells of human islets. Color scheme is the same as Fig 4.
Mentions: Calcium imaging during multiple glucose challenges was performed on intact human islets which were subsequently stained for marker expression (Fig 5). Population and single cell calcium imaging of human islets show responses to high glucose but with less synchronized responses compared to that of WT mouse islets (Fig 5A and 5B, 5C and S9 Movie). Similar to WT mouse islets, human islets that responded to all 3 high glucose stimulations were mostly β cells, as determined by INS/NKX6.1 coexpression. Cells that did not respond to high glucose showed an inversed calcium response to low glucose and expressed α cell markers (Fig 5D, 5E, and 5F). Human islets also contained dysfunctional β cells that showed partial calcium responses to glucose challenges with lower calcium influx peaks compared to fully calcium responsive cells (Fig 5F). Unlike WT mouse islets, human islets contained cells expressing INS marker alone and these showed three significant responses to high glucose challenges, albeit with lower calcium peaks than the response of double positive, INS/NKX6.1 human β cells.

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.