CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells

Animal procedures were approved by the local ethics committee for use of laboratory animals in Malmö, Sweden. Human islet isolation from deceased donors and experimental protocols were approved by the ethics committee in Uppsala and Malmö, Sweden.

Islets from 43 non-diabetic human donors (F/M 18/25, age 59 ± 1.3, body mass index (BMI) 25.5 ± 0.5 kg/m², HbA1c 5.7 ± 0.04, days in culture 4.1 ± 0.3) were used for secretion measurements, patch-clamp experiments, qPCR and immunohistochemistry. For these experiments islets were hand-picked to ensure high purity. Human islets were provided by the Nordic Network for Clinical Islet Transplantation (Uppsala, Sweden) through the LUDC Human Tissue Laboratory. Female NMRI mice (Bolmholtgaard, Ry, Denmark) were sacrificed by cervical dislocation and islets were isolated by collagenase digestion and hand-picked prior to experiments. For patch-clamp measurements, human and mouse islets were dispersed into single cells as previously described [19].

Total RNA from human and mouse islets were prepared as described [20]. CFTR mRNA expression was measured by RT-qPCR using primers and probes from Taqman mRNA assays (Life Technologies, California, USA). Human CFTR: Hs00357011_m1 expression was normalized against human HPRT1: 4333768 F, while mouse CFTR: Mm00445197_m1 expression was normalized against mouse HPRT1: Mm00446968. RT-qPCR runs were performed for individual batches of human (N = 5) and mouse (N = 5) islets in triplicate wells of 384-well plate on a 7900 HT RT-PCR system (Applied Biosystems, California, USA).

Human or mouse single cells were fixed and stained as described elsewhere [21]. Primary antibodies; Mouse monoclonal anti-CFTR (MATG-1061, RD-Biotech, France), Guinea pig monoclonal anti-insulin (Linco, Billerica, MA, USA), Guinea pig monoclonal anti-insulin (EuroDiagnostica, Malmö, Sweden) and rabbit polyclonal anti-Stx1A (Synaptic Systems, Goettingen, Germany). Secondary antibodies; guinea pig conjugated to Cy2, mouse conjugated to Cy3, guinea pig and rabbit conjugated to Cy5 (all from Jackson, UK). Immunofluorescence was detected with a confocal microscope (META 510, Zeiss, Germany) and unspecific binding of the secondary antibodies was excluded by parallel experiments in the absence of the primary antibodies. Localization of CFTR was analyzed as described elsewhere [22]. In short, the ratio between the mean fluorescent intensity in the plasma membrane region (P₁) and the cytosolic region (P₂) was measured using ZEN software (Zeiss, Jena, Germany). Pixel by pixel co-localization analysis was also performed using Zen software. The same laser setting was retained between experiments enabling comparison of experiments performed at different occasions.

EPC10 amplifier and 8.80 pulse software (HEKA, Lambrecht/Pfalz, Germany) was used to evoke and record whole-cell currents and changes in membrane capacitance on single mouse and human islet cells as described [19]. Current and capacitance measurements were performed at RT and 32 to 33°C, respectively. Extracellular solution contained: 118 mM NaCl, 20 mM TEACl, 5.6 mM KCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 5 mM HEPES, 3 (current measurements)/5 (capacitance measurements) mM D-glucose pH 7.4 (NaOH), supplemented with 10 μM FSK, 200 μM 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), 10 μM CFTR_inh-172 (Sigma Aldrich, Sweden), 25 μM (current measurements) or 40 μM (capacitance measurements) GlyH-101 (Calbiochem, USA) [23, 24] and 50 μM T16Ainh-AO1 (gift from A.S. Verkman, Department of Medicine, UCSF School of Medicine) as indicated. The intracellular solution contained: 125 mM CsOH, 125 mM Glutamate, 10 mM CsCl, 10 mM NaCl, 1 mM MgCl₂, 3 mM Mg-ATP, 4 mM EGTA (current measurement)/0.05 mM EGTA (capacitance measurement), 5 mM HEPES and 0.1 mM cAMP (capacitance measurements) (pH 7.15 with CsOH).

Insulin secretion was measured in static batch incubations described previously [25]. Briefly islets were pre-incubated in 1 mM glucose for 30 minutes followed by 1 h incubation in extracellular solution with variable glucose concentration. Incubation was 15 min when 50 mM KCl was used as stimulator. The extracellular solution was supplemented with 10 μM FSK, 0.1 μM GLP-1 (Bachem, Weil am Rhein, Switzerland), 100 μM Tolbutamide (Sigma Aldrich, Stockholm, Sweden), 200 μM DIDS, 50 μM TMinh-AO1, 40 μM CFTRinh-172 and/or 50 μM GlyH-101 as indicated. Insulin secretion was measured using radioimmunoassay kit (mouse: Linco Research Inc.,; human: Millipore, Billerica, MA, USA).

Islets were collected after insulin secretion assay and prepared for electron microscopy, examined and analyzed as previously described [26]. The granule volume density (N_v) and surface density (N_s) were calculated using in-house software programmed in MatLab.

Data are presented as mean ± SEM of N number of individuals or independent experiments and number of cells or biological replicates. Statistical significance was calculated using ANOVA or Student’s t test and P <0.05 was considered significant.

To investigate if CFTR affects beta-cell function, the influence of CFTR on insulin secretion was measured from isolated human and mouse pancreatic islets subjected to glucose-induced insulin secretion assays. In human islets, glucose caused a dose-dependent increase in insulin secretion that was further enhanced by the cAMP-increasing agent FSK. The stimulatory action of FSK at 16.7 mM glucose was significantly reduced in the presence of the CFTR-antagonist GlyH-101 (Figure 1A). Next we investigated the effect of a more physiological elevator of cAMP, the incretin hormone GLP-1. Like FSK, GLP-1 enhanced insulin secretion at 16.7 mM glucose in human islets, an effect that was completely inhibited by GlyH-101 (Figure 1B). In mouse islets, both FSK and GLP-1 amplified insulin secretion stimulated by glucose was reduced in the presence of GlyH-101. Moreover, CFTRinh-172, another antagonist against CFTR, also decreased both FSK and GLP-1 enhanced insulin secretion (Figures 1C, D). Both inhibitors are specific for CFTR, and whereas CFTRinh-172 binds to the intracellular domain of CFTR [23] and thereby closes the channel, GlyH-101 is an open-channel blocker [24]. It was confirmed that the antagonists had no significant effect on insulin secretion at 1 mM glucose (Figure 1E) or in presence of 16.7 mM glucose in the absence of FSK or GLP-1 (Figure 1F). There was, though, a tendency towards a reduction in insulin secretion by GlyH-101 in the presence of 16.7 mM glucose alone in the human islets. This might be due to a higher increase in cAMP caused by glucose. It has been demonstrated that glucose increases cAMP levels in both mouse [27] and human [28] beta-cells. However, why the increase in cAMP generated by glucose might be higher in human islets is not clear but could be explained by the higher number of alpha-cells in the human islets [29, 30]. cAMP is a potent second messenger that promotes insulin secretion by several mechanisms [19, 31]. Here we establish that CFTR has an essential and complementary function in cAMP-amplified insulin secretion. In parallel to these findings, we found expression of CFTR mRNA (data not shown) and CFTR protein in both human and mouse islets (Figure 1G).

The presence of active CFTR channels in pancreatic beta-cells was investigated on single cells using the patch-clamp technique in the standard whole-cell configuration. The pipette solution contained sodium and calcium ions in order to determine the cell-type by sodium channel inactivation properties [32]. A voltage-ramp protocol from −100 mV to +100 mV was applied before and every fourth minute after the addition of FSK (10 μM) until steady state was achieved (Figure 2). In the absence of FSK the current flow was minimal, whereas the increase in intracellular cAMP induced by FSK activated a non-linear outward rectifying current. In human and mouse beta-cells, the cAMP-activated current was significantly inhibited by the CFTR-inhibitors (Figure 2A-D). The current inhibited by CFTR-inhibitors (CFTR-dependent) constitute 47 ± 15% (n = 7) and 57 ± 7% (n = 10) of the FSK-activated current at negative potentials, in human and mouse beta-cells, respectively.

In addition to the ion channel function, CFTR has been attributed a role as regulator of other ion channels and proteins, such as other chloride channels [2, 33]. To investigate the possibility that CFTR regulates the function of other chloride channels we used the non-specific chloride channel blocker DIDS that blocks a wide variety of chloride channels, while CFTR is insensitive to this antagonist [34, 35]. The cAMP-stimulated current, in human and mouse beta-cells, was significantly reduced by DIDS (Figure 2E, F). The presence of active CFTR channels was proven by a significant reduction in current conductance in the simultaneous presence of GlyH-101 and DIDS as compared with DIDS alone (Figure 2E, F). The DIDS-sensitive component constituted 38 ± 10% and CFTR 37 ± 15% (n = 5) of the FSK-activated current at negative potentials (−100 to −50 mV) in human beta-cells. In the mouse, the DIDS-sensitive current was 41 ± 12% and CFTR 29 ± 9% (n = 8) of the FSK-activated current. From these measurements the CFTR conductance was estimated to be 2.8 ± 0.6 pS/pF in human (n = 5; N = 3) and 12 ± 4.0 pS/pF in mouse (n = 8; N = 5) beta-cells at negative potentials (−100 mV to −50 mV). Taken together these data indicate that 1) the cAMP-induced chloride current has one DIDS-sensitive component and one part sensitive to CFTR inhibition, 2) the current through CFTR in beta-cells is small, and 3) CFTR likely acts as a regulator of DIDS-sensitive chloride channel(s).

The suggestion that CFTR can act as a regulator upstream of other chloride channels involved in the regulation of insulin release was verified through the investigation of the combined effect of DIDS and GlyH-101 on insulin secretion. The experiment was conducted in the presence of tolbutamide to circumvent that DIDS might affect the ATP-dependent potassium channel [36]. At high concentrations tolbutamide can inhibit CFTR currents [37], but at the concentration used here (100 μM) the major influence is on the closure of the ATP-dependent potassium channel. This was confirmed by insulin secretion measurements showing that tolbutamide increased (rather than decreased) insulin secretion at 16.7 mM glucose in mouse islets, while no further stimulation of insulin secretion was observed in human islets (Figure 3A, B). This is most likely due to the different sensitivity of glucose in mouse and human islets and in line with previous results [38, 39]. In the presence of tolbutamide, GLP-1-enhanced glucose-stimulated insulin secretion was significantly reduced in the presence of DIDS (Figure 3A, B), in both mouse and human islets. The addition of GlyH-101 in the continued presence of DIDS did not further reduce the secretory response in mouse beta-cells as compared to DIDS alone (Figure 3A), suggesting that CFTR regulates a DIDS-sensitive chloride channel.

Recently, it was suggested that channels from the family of Anoctamins (ANO1-10) are controlled by CFTR [40, 41]. The first member in this family, ANO1, is a voltage- and calcium-dependent chloride channel localized in the plasma membrane that has been suggested to interact with CFTR to control chloride conductance in epithelial cells [41]. Interestingly, ANO1 gene expression has been measured in rat and human islets [42, 43]. To investigate the possible combined involvement of ANO1 and CFTR in the regulation of insulin secretion we performed insulin secretion measurements in mouse and human islets in the presence or absence of specific blockers of the respective channel. Indeed, insulin secretion enhanced by GLP-1 was reduced by TM16Ainh-AO1 (AO1), a specific blocker of ANO1 [44], and in the combined presence of GlyH-101 and AO1 the reduction in GLP-1 enhanced glucose-stimulated insulin secretion was not significantly different from the reduction observed after application of either of them alone (Figure 3C, D). In human islets, the inhibitory effect of AO1 was significantly more potent than GlyH-101 alone, but, as in the mouse, inclusion of GlyH-101 in the simultaneous presence of AO1 did not cause an additive decrease in GLP-1 amplified insulin secretion at 16.7 mM glucose (Figure 3D). It was confirmed that AO1 did not have any inhibitory effect at 1 mM glucose (Figure 3E).

The presence of ANO1 chloride current in beta-cells was measured by patch-clamp recordings on single mouse beta-cells using the standard whole-cell mode and the ramp-protocol as used above. It was obvious that the FSK-activated current was reduced by AO1, and the conductance was decreased (Figure 3F). A small, but significant, further reduction in conductance was obtained in the simultaneous presence of AO1 and the CFTR-inhibitor GlyH-101 (Figure 3F). The AO1-sensitive current was calculated to represent 50 ± 10% and CFTR 30 ± 5% of the FSK-activated current at negative potentials (n = 5). The CFTR conductance was estimated to 14 ± 3 pS/pA (n = 5). Interestingly, these are similar to values obtained from the DIDS-experiments in Figure 2 (12 pS/PA and 29%). This experiment demonstrates that the blockers AO1 and GlyH-101 most likely act on separate channels (Figure 3E). Hence, the lack of an additive effect on secretion (Figure 3C, D) would suggest that CFTR interacts with ANO1 in regulating insulin secretion.

We were next interested in investigating mechanisms by which CFTR and ANO1 influence insulin secretion. Insulin secretion comprises a cascade of events often referred to as the “stimulus-secretion-coupling” [45]. Briefly, glucose uptake and metabolism yields ATP, leading to depolarization of the plasma membrane, opening of voltage sensitive calcium channels and increased levels of intracellular calcium resulting in exocytosis of insulin granules. Theoretically, CFTR could be involved in any of these steps. Indeed, it has been suggested that an ATP-sensitive and cAMP-activated chloride current influences the membrane depolarization and electrical activity of insulin secreting cells [17]. We found that CFTR-inhibition reduced insulin secretion under conditions independent of cellular metabolism and membrane depolarization (Figure 4A), suggesting a possible function of CFTR in the downstream exocytotic process. To elaborate further on this, capacitance recordings were performed on single beta-cells in the presence of ATP and cAMP in the intracellular solution. Exocytosis, evoked by a train of 10 500-ms depolarizations from −70 mV to 0 mV, was significantly reduced in human beta-cells pre-incubated with GlyH-101 (Figure 4B, C). The reduction in membrane capacitance increase was most prominent during the first two depolarizations reflecting rapid exocytosis of primed granules [46]. The exocytotic response in mouse beta-cells was likewise reduced after CFTR-inhibition (Figure 4D, E). Next, effects on voltage-dependent calcium currents were investigated. The current was evoked by 50-ms depolarizations from −70 mV to voltages between -50 and +50 mV in single human or mouse beta-cells and in the presence and absence of GlyH-101 or CFTRinh-172, respectively (Figure 4F, G). The intracellular solution was supplemented with cAMP to activate CFTR. The currents comprise a rapid sodium current followed by a more slowly opening calcium current. The charge was measured as the integral of the current and reflected the influx through the calcium channels. From the data we could conclude that the calcium current was not affected by CFTR inhibition (Figure 4F, G), suggesting a direct effect on the exocytotic machinery [21]. Interestingly, DIDS has earlier been demonstrated to reduce insulin exocytosis, an effect that has been coupled to intragranular ClC3 chloride channels and priming of the insulin granules [47], implying that CFTR can act by controlling ANO1 to influence priming and exocytosis. Depolarization-evoked exocytosis of primed granules is hypothesized to correspond to first phase insulin secretion [46], thus suggesting an important role of CFTR at this stage. Indeed, patients with CF exhibit reduced first phase insulin secretion [9, 15, 16].

The above results prompted us to investigate granular docking using TEM, since docking of the granules to the plasma membrane is vital for exocytosis. Mouse islets were subject to incubation in 1 mM glucose, 16.7 mM glucose in the absence and/or presence of FSK and GlyH-101 prior to fixation (Figure 5A). We performed ultrastructural analysis and estimated the granule volume density per cell, N_v, and the surface density per cell, Ns, from the micrographs. N_v and N_s are proportional to the total number of granules and the number of docked granules, respectively. The total number of granules within the beta-cells was not changed between islets treated under the different conditions (Figure 5B). Glucose significantly increased the docked pool (N_s) in agreement with previous observations that glucose enhances granule refilling and mobilization [48]. Addition of FSK caused a reduction in the number of docked granules, confirming earlier electrophysiological observations that mobilization is rate-limiting [49]. Finally, the docked pool of granules was significantly reduced in beta-cells incubated with GlyH-101 and FSK, as compared to FSK alone (Figure 5C). Moreover, it is evident from the analysis that only the fraction of granules within 300 nm from the plasma membrane is affected by inhibition of CFTR (Figure 5D). This is in accordance with a role for CFTR in docking and priming of insulin granules.

CFTR has been shown to act through additional mechanisms, one being interaction with the SNARE-protein syntaxin 1A [50, 51], a protein crucial for beta-cell granule docking and exocytosis [46]. To investigate the possibility that CFTR interacts with this SNARE-protein, we investigated the distribution of CFTR and syntaxin 1A, and found that CFTR was co-localized with syntaxin 1A in the plasma membrane of human beta-cells (86 ± 2% co-localization, n = 39; N = 2; Figure 5E).