Fig 1.
A rise in intracellular calcium corresponds to insulin secretion.
(A) Overview of calcium imaging method for islets: intact islets were plated on a 96 well plate. Islets were stained with the Fluo-4 AM and then washed in fasting solution. Calcium influx of the population of cells in the islet or single cells was imaged following the addition of glucose. Same islets were imaged again after fixation and immunofluorescence staining with INS, GCG, and NKX6.1. (B) Representative images of WT mouse islets after stimulating with 2.5 and 15 mM glucose followed by 30 mM KCl. (C) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity for WT mouse islets shown in Fig 1B (7 islets) with corresponding S1 Movie. The calcium influx response for each mouse islet was normalized to the starting fluorescence intensity data point during the initial low glucose incubation. The normalized fluorescence intensities of 7 WT mouse islets were averaged and plotted on the y-axis with standard deviations. Islets were challenged sequentially with 2.5, 15, 2.5, 15, 2.5, and 15 mM glucose and 30 mM KCl. Fluorescence was measured at 126 time points throughout the series of glucose challenges, normalized to the starting fluorescence intensity, and averaged across all islets at each time point. The standard deviation at each time point ranged between ± 2 to 8 a.u.c (area under the curve). The x-axis represents time (in seconds). P-value was calculated from the difference between ave a.u.c. during low glucose and high glucose stimulations. Significance of calcium influx response was labeled * when the P-value was between 0.05 and 0.001 and ** when the P-value was below 0.001. (D) Average normalized population measurements with standard deviation of dynamic Fluo-4 fluorescence intensity of a total of eight WT mouse islets with corresponding S2 Movie. Population measurements of dynamic normalized Fluo-4 fluorescence intensity for mouse islets is shown in purple, and the ELISA measurements of secreted mouse insulin for the same batch of islets is shown in blue. Challenges were done with 5 minutes of 2.5 mM, 25 minutes of 15 mM, and 5 minutes of 30 mM KCl.
Fig 2.
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.
Fig 3.
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.
Fig 4.
Analysis of islet cell calcium response with staining for endocrine markers allows further characterization of WT and diabetic islets.
Color scheme for the list of markers analyzed is on the left panel; INS/NKX6.1 (Purple), NKX6.1 (Blue), INS (Red), and GCG (Green). Pie charts on the right panel show the composition of marker expression with the indicated frequency of glucose responsiveness for (A) WT, (B) db/db, and (C) NOD mouse islets. Average normalized single cell measurements with standard deviation of dynamic Fluo-4 fluorescence intensity on the left graphs. Single cell measurements of dynamic Fluo-4 fluorescence intensity on the right graphs.
Fig 5.
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.