Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes

Figures

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  FIGURE 1. NOD mice are normoglycemic at 12 weeks of age. (A) Blood glucose levels in 12-week old NOD (n = 7–13) and NOD-RAG1KO (n = 8–15) mice following a glucose tolerance test starting at time 0. (B) Glucose area under the curve for 6, 9, and 12 week old NOD (red) and NOD-RAG1KO (black) mice calculated from blood glucose levels measured over 2 h following glucose challenge in as A. (C) Insulin secretion normalized to insulin content in isolated islets from 6, 9, and 12 week old NOD (n = 4–6) and NOD-RAG1KO (n = 3–5) mice at 20 mM glucose. Data in all panels represents the mean ± SEM. In B and C, data from individual mice are represented by black and red squares for NOD-RAG1KO and NOD mice respectively. All animals are female unless otherwise noted.

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  FIGURE 2. NOD mice have altered Ca2+ signaling as early as 9 weeks of age. Representative plots of intracellular Ca2+ as measured by fluorescence intensity at 11 mM glucose in 5 individual cells in the same islet over time in 9-week old (A) NOD-RAG1KO (black, n = 3–4) and (B) NOD (red, n = 3–5) mice. Quantification of the fraction of the islet area showing intracellular Ca2+ activity at (C) 2 mM glucose, (D) 5 mM glucose, and (E) 11 mM glucose in isolated islets from 6, 9, and 12-week old NOD and NOD-RAG1KO mice. (F) Quantification of the fraction of the islet area with coordinated Ca2+ oscillations at 11 mM glucose in isolated islets from 6, 9, and 12-week old NOD and NOD-RAG1KO mice. Data in C-F represents the mean ± SEM. Data from individual mice are represented by black and red squares for NOD-RAG1KO and NOD mice respectively (n = 3–6). p < 0.05 is significant as determined by ANOVA. All animals are female unless otherwise noted.

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  FIGURE 3. NOD mice have decreased β-cell coupling as early as 9 weeks of age and decreased β-cell coupling in the NOD mouse is mediated by autoreactive T-cells. (A) β-cell coupling as measured by fluorescence recovery rate in isolated islets from 6, 9, and 12-week old NOD (red, n = 6–9) and NOD-RAG1KO (black, n = 5–6) mice. Data represent the mean ± SEM. Data from individual mice are represented by black and red squares for NOD-RAG1KO and NOD mice respectively (n = 3–6). p < 0.05 is significant as determined by ANOVA. (B) K-means clustering analysis of the correlation between β-cell coupling and the fraction of the islet area with coordinated intracellular Ca2+ oscillations at 11 mM glucose in NOD and NOD-RAG1KO mice at 9 weeks of age. Red solid and dashed ovals indicate the clusters of animals identified by k-means analysis. (C) β-cell coupling as measured by fluorescence intensity recovery rate in isolated islets from 12-week old NOD (red), NOD-RAG1KO (black), and NOD chromogranin-A knockout (NOD-ChgA KO, green). Data from individual mice are represented by black, red, or green squares for NOD-RAG1KO, NOD, or NOD-ChgA KO mice respectively (n = 4–5). (D) β-cell coupling as measured by…

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  FIGURE 4. Modulation of Cx36 Coupling and Intracellular Ca2+ Mediate Cytokine-Induced Islet Death. (A) β-cell coupling rate as measured by FRAP in islets from WT and RIP Cx36 mice treated with 0, 0.01, or 0.1 relative cytokine concentration (RCC) for 24 h (n = 5). (B) Percent cell death islets from WT and RIP Cx36 mice treated with or without cytokines (1 RCC) and with or without the K+ channel inhibitor TEA for 24 h (n = 3–5). Percent cell death in islets from WT and Cx36 KO mice treated with or without (C) 250 nM of the KATP channel activator Diazoxide (n = 5) or (D) 20 mM of the K+ channel inhibitor TEA (n = 3) with or without a cytokine cocktail and for 24 h. Percent cell death in WT and Cx36 KO islets treated with or without (E) 1 µM of the SERCA activator Ochratoxin A (n = 5) or (F) 1 µM of the SERCA inhibitor thapsigargin (n = 5) with or without cytokines (1 RCC) for 24 h. Data represent the mean ± SEM. p < 0.05 is significant as determined by ANOVA. * Indicates p < 0.05 compared to untreated controls in A.

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  FIGURE 5. Overexpression of Cx36 in the NOD mouse. (A) Gene expression of Cx36 in 8–12-week old male WT NOD and RIPCx36-NOD mice normalized to HPRT1 housekeeping gene using the ΔΔCt method (n = 4). (B) β-cell coupling as measured by fluorescence recovery rate in isolated islets from 12-week old male WT NOD (grey) and RIPCx36-NOD (blue) mice (n = 4). (C) Gene expression of Cx36 in 12-week old female WT NOD and RIPCx36-NOD mice normalized to HPRT1 housekeeping gene using the ΔΔCt method (n = 3). (D) β-cell coupling as measured by fluorescence intensity recovery rate in isolated islets from WT NOD and RIPCx36-NOD mice (n = 4–5). Quantification of the fraction of the islet area showing intracellular Ca2+ activity (E) and the fraction of the islet area with coordinated Ca2+ oscillations (F) at 11 mM glucose in isolated islets from 12 week old WT NOD and RIPCx36-NOD mice (n = 3–4). Representative plots of intracellular Ca2+ as measured by fluorescence intensity at 11 mM glucose in 5 individual cells in the same islet over time in 12-week old (G) WT NOD (grey) and (H) RIPCx36-NOD (blue) mice. Data from individual mice are represented by grey or blue squares for WT NOD or RIPCx36-NOD mice…

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  FIGURE 6. Overexpression of Cx36 protects against T1D onset between 10–16 weeks of age in the NOD mouse. (A) Kaplan-Meier survival analysis of diabetes onset in WT NOD (grey, n = 34) and RIPCx36-NOD (blue, n = 38) mice. p < 0.05 indicates a significant difference in survival probability as determined by the Breslow test. (B) Kaplan-Meier survival analysis of diabetes onset in NOD mice (NOD colony, red, n = 77) and RIPCx36-NOD (blue, n = 38) mice. p < 0.05 indicates a significant difference in survival probability as determined by the Breslow test. (C) Average islet mass per pancreas in 12-week old WT NOD (grey) and RIPCx36-NOD (blue) pancreata. Representative images of H&E staining in WT NOD (D) insulitis score = 2 and RIPCx36-NOD (E) insulitis score = 3 pancreas. (F) Fraction of total islets averaged over 6 slices per pancreas with an insulitis score of 0, 1, 2, or 3 in 12-week old WT NOD (n = 5) and RIPCx36-NOD (n = 6) mice as determined by IHC. Representative images of TUNEL and insulin staining in pancreas sections from WT NOD (G) and RIPCx36-NOD (H) mice. Red arrows indicate TUNEL+ insulin + cells. (I) Histogram of the average fraction of total islets with increasing percentage of…