Glucose-induced electrical activities and insulin secretion in pancreatic islet β-cells are modulated by CFTR

Jing Hui Guo, Hui Chen, Ye Chun Ruan, Xue Lian Zhang, Xiao Hu Zhang, Kin Lam Fok, Lai Ling Tsang, Mei Kuen Yu, Wen Qing Huang, Xiao Sun, Yiu Wa Chung, Xiaohua Jiang, Yoshiro Sohma, Hsiao Chang Chan, Jing Hui Guo, Hui Chen, Ye Chun Ruan, Xue Lian Zhang, Xiao Hu Zhang, Kin Lam Fok, Lai Ling Tsang, Mei Kuen Yu, Wen Qing Huang, Xiao Sun, Yiu Wa Chung, Xiaohua Jiang, Yoshiro Sohma, Hsiao Chang Chan

Abstract

The cause of insulin insufficiency remains unknown in many diabetic cases. Up to 50% adult patients with cystic fibrosis (CF), a disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), develop CF-related diabetes (CFRD) with most patients exhibiting insulin insufficiency. Here we show that CFTR is a regulator of glucose-dependent electrical acitivities and insulin secretion in β-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca(2+) oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse β-cells or RINm5F β-cell line, or significantly attenuated in CFTR mutant (DF508) mice compared with wild-type mice. VX-809, a newly discovered corrector of DF508 mutation, successfully rescues the defects in DF508 β-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in β-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

Figures

Figure 1. CFTR Cl − currents in…
Figure 1. CFTR Cl− currents in mouse pancreatic islet β-cells and its activation by glucose.
(a,b) Whole-cell Cl− currents recorded with CsCl pipette solution in CFTR wild-type (a) or DF508 mutant (b) β-cells before (control) and 5 min after the addition of forskolin (10 μM) and 3 min after CFTR inhibitor glyH-101 (10 μM) with corresponding I-V curves (n=3). (c) Whole-cell Cl− current recorded with NMDG-Cl pipette and bath solution in wild-type mouse β-cells before (control) and 10 min after the addition of glucose (10 mM), and subsequently 3 min after glyH-101 (10 μM) with corresponding I-V curves (n=3). Pulse protocol: 20 mV steps from −100 mV to +100 mV for 100 ms. Extracellular Cl− concentration ([Cl−]o)=142 mM; Intracellular Cl− concentration ( [Cl−]i )=150 mM; equilibrium potential for Cl− (ECl)=1.4 mV. Data are shown as mean±s.e.m.
Figure 2. Involvement of CFTR-medicated Cl −…
Figure 2. Involvement of CFTR-medicated Cl− efflux in maintaining resting membrane potential of β-cells.
(a) CFTR inhibitors, CFTRinh-172 (10 μM) and glyH-101 (10 μM), induced membrane hyperpolarization in RINm5F cells assessed by voltage-sensitive fluorometric measurement with Dibac on RINm5F cells. Number of measurements is shown in each column. (b) Intracellular chloride measurement with MQAE chloride-sensitive fluorescent dye in RINm5F cells (left panel: calibration curve, right panel: time course of changes in [Cl−]i). Application of CFTRinh-172 (10 μM) led to elevation of [Cl−]i by 25.9±1.3 mM (n=36). (c) Membrane potential measurement by patch-clamp in RINm5F cells. CFTRinh-172 (10 μM) led to hyperpolarization of the membrane. **P<0.01, t-test. The experiment was repeated five times. (d) Membrane potential of β-cells measured by patch-clamp. Freshly isolated β-cells from DF508 mutant mice (n=4) had more negative resting membrane potential than that from wild-type mice (n=5). *P<0.05, t-test. (e) KATP channel inhibitor glibenclamide (GLIB, 1 μM) depolarized the membrane (left), whereas in the presence of glyH-101 (10 μM), GLIB failed to induce depolarization (right) in RINm5F cells. Number of measurements is shown in each column of the summary chart. ***P<0.001, one-way analysis of variance (ANOVA). (f) Glucose (10 mM) induced depolarization by about 20 mV in RINm5F cells. Pretreatment with CFTRinh-172 (10 μM) and glyH-101 (10 μM) inhibited the glucose-induced depolarization. Number of measurements is shown in each column. ***P<0.001, one-way ANOVA. (g,h) Recording of action potentials evoked by current injection in RINm5F cells; (g) Knockdown of CFTR (CFTRkd) decreased the action potential evoked by 0.05 and 0.3 nA currents, as compared with LacZ control, with number of experiments shown in data bars. *P<0.05, t-test. Western blots show the protein level of CFTR after knockdown. Uncropped immunblot is shown in Supplementary Fig. 8. (h) CFTRinh-172 (10 μM) completely abolished the action potential evoked by 0.05 nA current stimulus and partially abolished the action potential evoked by 0.3 nA current stimulus with number of experiments shown in corresponding data bars. *P<0.05, **P<0.01, t-test. [Cl−]o=142 mM (a–h). [Cl−]i=98 (a–f) and 150 (g,h) mM. ECl= −9.8 (a–f) and 1.4 (g and h). Data are shown as mean±s.e.m.
Figure 3. Involvement of CFTR-mediated Cl −…
Figure 3. Involvement of CFTR-mediated Cl− efflux in glucose-induced electric spikes and calcium oscillations in β-cells.
(a) Glucose (10 mM) elicited a slow membrane depolarization superimposed with bursts of action potentials (spikes) in isolated mouse islet β-cells (enlarged in I), which could be abolished by CFTRinh-172 (10 μM) (enlarged in II). n=4. (b) Islet β-cells from DF508 mice required a significantly longer time to reach the threshold for spike generation and had a smaller magnitude and frequency of glucose (10 mM)-induced spikes than that from the wild types. Number of measurements is shown in each column *P<0.05, ***P<0.001, t-test. (c) When the membrane potential was clamped to −65 mV similar to that observed in wild-type, DF508 β-cells (lower panel) still exhibited a reduced magnitude of the glucose-induced spike than that from wild type (upper panel). Number of measurements is shown in each column. *P<0.05, t-test. (d) Upper panel: when the membrane potential was clamped to depolarization voltage (−40 mV), electric spikes generated in wild-type β-cells exhibited higher magnitude and frequency than that in DF508. Lower panel: when the membrane potential was elevated to a depolarized level, −50 mV, CFTRinh-172 (10 μM) decreased the spike frequency and amplitude in RINm5F cells. Number of measurements is shown in each column. *P<0.05, ***P<0.001, t-test. [Cl−]o=142 mM; [Cl−]i=150 mM and ECl=1.41 mV (a–d). (e) Intracellular chloride concentration-dependent action potential in RINm5F cells. [Cl−]o=142 mM; ECl=1.4, −16.5 and −∞ with [Cl−]i at 150, 75 and 0 mM, respectively. n=4. **P<0.01; ***P<0.001, one-way analysis of variance (ANOVA). (f) When Cl− was removed from the pipette solution, the glucose-induced electric spikes were almost completely abolished in RINm5F cells. (g) Effect of CFTRinh-172 (10 μM) and glyH-101 (10 μM) on glucose (10 mM)-induced intracellular Ca2+ and oscillations measured by Fura-2 in RINm5F cells. Number of measurements is shown in each column. ***P<0.001, one-way ANOVA. (h) Glucose (10 mM)-induced Ca2+ increase and oscillations in β cells isolated from the wild-type and DF508 mice with or without CFTRinh-172 (10 μM). Number of measurements is shown in each column. ***P<0.001, one-way ANOVA. Data are shown as mean±s.e.m.
Figure 4. Involvement of CFTR in glucose-induced…
Figure 4. Involvement of CFTR in glucose-induced insulin secretion.
(a) Effect of CFTRinh-172 and glyH-101 on glucose-induced insulin secretion of islets isolated from mice. CFTRinh-172 (10 μM) and glyH-101 (10 μM) significantly reduced insulin secretion 5, 15 and 60 min after glucose (10 mM) challenge. The experiment was repeated three times. *P<0.05, ***P<0.001, one-way analysis of variance. (b) Reduced glucose (10 mM) -induced insulin secretion from CFTR DF508 islets as compared with the wild-type. n=3–4, *P<0.05, t-test. (c,d) Time-course change in blood glucose (c) and insulin (d) after glucose injection (intraperitoneal 2 g kg−1 body weight) in DF508 and the wild-type mice. *P<0.05; **P<0.01. n=6, t-test. Data are shown as mean±s.e.m.
Figure 5. Rescue of DF508 defects by…
Figure 5. Rescue of DF508 defects by corrector VX-809.
Effect of VX-809, a corrector of DF508 CFTR, in rescuing DF508 defects, resting membrane potential (a, by patch-clamp), Ca2+ response (b, by Fura-2) and insulin secretion (c). Islets/β-cells isolated from DF508 mice were pretreated with VX-809 (10 μM) for 48 h before the experiments. DMSO (0.1% v/v) was also applied to both wild-type and DF508 islets/β-cells as vehicle control for comparison. Number of measurements is shown in each column. *P<0.05,**P<0.01, one-way analysis of variance. Data are shown as mean±s.e.m.

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