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, 8 (1), 1158

Cytokine-mediated Changes in K + Channel Activity Promotes an Adaptive Ca 2+ Response That Sustains β-Cell Insulin Secretion During Inflammation

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Cytokine-mediated Changes in K + Channel Activity Promotes an Adaptive Ca 2+ Response That Sustains β-Cell Insulin Secretion During Inflammation

Matthew T Dickerson et al. Sci Rep.

Abstract

Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca2+ influx (GSCI) and endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) handling, leading to diminished glucose-stimulated insulin secretion (GSIS). However, cytokine-mediated changes in ion channel activity that alter β-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated β-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered β-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca2+ content to maintain GSIS.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cytokine exposure reduces islet KCNK16 transcript abundance and associated TALK-1 protein. (a) qRT-PCR analysis of KCNK16 (encodes TALK-1) and ATP2A2 (encodes SERCA2b) transcript relative to GAPDH in nontreated (gray) and cytokine treated (black) WT mouse islets (N = 4 animals), (b) western blot analysis of human islet TALK-1 protein content with (black) and without (gray) cytokines (N = 3 donors), (c) images of human islet TALK-1 western blots for all donors, (d) representative immunofluorescent images of nontreated (upper panels) and cytokine treated (lower panels) mouse islet slices (TALK-1 - green, insulin - red, and nucleus - blue; scale bars are 20 µm), (e) average TALK-1 fluorescence intensity in nontreated (gray) and cytokine treated (black) mouse islet slices (N ≥ 3 islet slices), (f) representative immunofluorescent images of nontreated (upper panels) and cytokine treated (lower panels) human islet slices, and (g) average TALK-1 fluorescence intensity in nontreated (gray) and cytokine treated (black) human islet slices (N ≥ 5 islet slices). Statistical analysis was conducted using unpaired two-tailed t-tests and uncertainty is expressed as SEM (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 2
Figure 2
Cytokine exposure elevates basal islet [Ca2+]i and disrupts GSCI. (a) Representative Fura-2 AM recordings (F340/F380) of changes in β-cell [Ca2+]i for nontreated (gray) and cytokine treated (black) WT mouse islets (the lines above the figure indicate glucose concentrations), (b) average [Ca2+]i oscillation frequency for nontreated (gray) and cytokine treated (black) WT mouse islets with 11 mM glucose (N ≥ 12 islets), (c) representative Fura-2 AM recordings of changes in β-cell [Ca2+]i for nontreated (gray) and cytokine treated (black) TALK-1 KO mouse islets, (d) average [Ca2+]i oscillation frequency for nontreated (gray) and cytokine treated (black) TALK-1 KO mouse islets with 11 mM glucose (N ≥ 30 islets), (e) average basal [Ca2+]i AUC (0–200 sec) for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO islets (N = 3 animals), (f) average 1st phase GSCI AUC (200–600 sec) for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO islets (N = 3 animals), and (g) average 2nd phase GSCI AUC (600–1000 sec) for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO islets (N = 3 animals). Statistical analysis was conducted using 1-way ANOVA and uncertainty is expressed as SEM (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 3
Figure 3
Cytokine exposure increases β-cell [Ca2+]ER. (a) Representative Fura-2 AM CPA responses of nontreated (gray) and cytokine treated (black) WT mouse β-cells (left; black bars correspond to the addition of CPA) and relative cytokine-induced change in [Ca2+]i AUC (right; N = 3 animals), (b) representative Fura-2 AM CPA responses of nontreated (gray) and cytokine treated (black) TALK-1 KO mouse β-cells (left) and relative cytokine-induced change in [Ca2+]i AUC (right; N = 3 animals), (c) average peak CPA responses ([F340/380]max/F[340/380]min) for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO mouse β-cells (N = 3 animals), (d) representative fluorescent images of the D4ER genetically encoded, β-cell specific [Ca2+]ER indicator in WT (top) and TALK-1 KO (bottom) islets (green: F535, blue: F475, cyan: merge; scale bar is 50 µm), and (e) average D4ER intensity (F535/F475) of WT and TALK-1 KO mouse β-cells from intact islets (N = 4 animals). Statistical analysis was conducted using 1-way ANOVA and uncertainty is expressed as SEM (*P < 0.05, **P < 0.01).
Figure 4
Figure 4
Cytokine exposure decreases TALK-1 K2P currents. (a) Representative whole-cell voltage-clamp recordings of K2P currents in nontreated (gray) and cytokine treated (black) WT mouse β-cells, (b) average K2P currents in nontreated (gray) and cytokine treated (black) WT mouse β-cells at selected voltages (N = 4 animals), (c) representative whole cell voltage ramp recordings of K2P currents in nontreated (gray) and cytokine treated (black) TALK-1 KO mouse β-cells, and (d) average K2P currents in nontreated (gray) and cytokine treated (black) TALK-1 KO mouse β-cells at selected voltages (N = 3 animals). Statistical analysis was conducted using paired two-tailed t-tests and uncertainty expressed as SEM (*P < 0.05, **P < 0.01).
Figure 5
Figure 5
Cytokine exposure increases β-cell electrical excitability. (a) Representative perforated-patch current-clamp Vm recording of a nontreated WT β-cell (lines above figure indicate glucose concentrations), (b) representative Vm recording of a cytokine treated WT β-cell, (c) representative Vm recording of a nontreated TALK-1 KO β-cell, (d) representative Vm recording of a cytokine treated TALK-1 KO β-cell, (e) average electrical oscillation frequency for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO β-cells (N ≥ 6 cells), (f) average resting Vm for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO β-cells (N ≥ 8 cells), (g) average interburst Vm for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO β-cells (N ≥ 7 cells), (h) average plateau Vm for nontreated (gray) and cytokine treated (black) WT and TALK-1 KO β-cells (N ≥ 8 cells). Statistical analysis was conducted using 1-way ANOVA and uncertainty is expressed as SEM (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 6
Figure 6
Cytokine exposure reduces β-cell Kslow currents. (a) Overview of Kslow perforated-patch voltage-clamp recording protocol, (b) representative nontreated WT β-cell Kslow trace (average of 6 cells from one animal), (c) representative cytokine treated WT Kslow trace (average of 6 cells from one animal), (d) average Kslow currents for nontreated WT (dark green) and TALK-1 KO (red) β-cells (N ≥ 15 cells), and (e) average Kslow currents for nontreated (dark green) and cytokine treated (light green) WT β-cells (N ≥ 31 cells).
Figure 7
Figure 7
Cytokine exposure decreases insulin protein and related transcripts in WT and TALK-1 KO islets but only disrupts GSIS from TALK-1 KO islets. (a) Average basal (2 mM glucose) and glucose-stimulated (7 and 11 mM glucose) insulin secretion from nontreated and cytokine treated WT and TALK-1 KO islets (N ≥ 3 animals), (b) average insulin content of nontreated (gray) and cytokine treated (black) WT and TALK-1 KO islets (N = 3 animals), and (c) preproinsulin (Ins1 and Ins2) and proprotein convertase 1 (PCSK1) transcript relative to GAPDH in nontreated and cytokine treated WT and TALK-1 KO islets (N = 3 animals). Statistical analysis was conducted using 1-way ANOVA and uncertainty is expressed as SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

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