Calcium influx activates adenylyl cyclase 8 for sustained insulin secretion in rat pancreatic beta cells
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Insulin is a key metabolic regulator in health and diabetes. In pancreatic beta cells, insulin release is regulated by the major second messengers Ca2+ and cAMP: exocytosis is triggered by Ca2+ and mediated by the cAMP/protein kinase A (PKA) signalling pathway. However, the causal link between these two processes in primary beta cells remains undefined.
Time-resolved confocal imaging of fluorescence resonance energy transfer signals was performed to visualise PKA activity, and combined membrane capacitance recordings were used to monitor insulin secretion from patch-clamped rat beta cells.
Membrane depolarisation-induced Ca2+ influx caused an increase in cytosolic PKA activity via activating a Ca2+-sensitive adenylyl cyclase 8 (ADCY8) subpool. Glucose stimulation triggered coupled Ca2+ oscillations and PKA activation. ADCY8 knockdown significantly reduced the level of depolarisation-evoked PKA activation and impaired replenishment of the readily releasable vesicle pool. Pharmacological inhibition of PKA by two inhibitors reduced depolarisation-induced PKA activation to a similar extent and reduced the capacity for sustained vesicle exocytosis and insulin release.
Our findings suggest that depolarisation-induced Ca2+ influx plays dual roles in regulating exocytosis in rat pancreatic beta cells by triggering vesicle fusion and replenishing the vesicle pool to support sustained insulin release. Therefore, Ca2+ influx may be important for glucose-stimulated insulin secretion.
KeywordsAdenylyl cyclase 8 Ca2+ Pancreatic beta cell Protein kinase A Vesicle pool
Normalised FRET ratio
Intracellular free Ca2+ concentration
Intracellular free cyclic AMP concentration
Cyan fluorescent protein
Exchange protein directly activated by cAMP 2
Fluorescence resonance energy transfer
Immediately releasable pool
Protein kinase A
Readily releasable pool
ADCY8 short hairpin RNA
Scrambled control short hairpin RNA
Yellow fluorescent protein
Voltage-gated Ca2+ channel
Insulin regulates blood glucose metabolism and helps maintain energy homeostasis. Impaired insulin secretion leads to glucose intolerance and diabetes. In response to increased blood glucose, the cytosolic ATP/ADP ratio in pancreatic beta cells increases . This change leads to the closure of KATP channels, which depolarises the membrane, causing Ca2+ influx and subsequent exocytosis of insulin granules [2, 3]. Thus, elevated intracellular Ca2+ ([Ca2+]i) in pancreatic beta cells is known to be the major trigger for glucose-stimulated insulin secretion.
Cyclic AMP (cAMP) is another important second messenger that enhances vesicle fusion via both protein kinase A (PKA)-dependent and PKA-independent pathways, which are dependent on exchange protein directly activated by cAMP 2 (EPAC2) [4, 5, 6]. Endogenous incretin hormones, such as glucose-dependent insulinotropic polypeptide  and glucagon-like peptide1 (GLP-1) , act on membrane receptors to generate cytoplasmic cAMP and regulate insulin secretion. Glucose metabolism also triggers cytoplasmic cAMP elevation and oscillations in pancreatic beta cells, although the underlying mechanisms and the physiological significance of this process remain unclear [9, 10, 11, 12].
Crosstalk between these key regulators of insulin secretion, the Ca2+ and cAMP signalling pathways, occurs within individual beta cells. Both Ca2+-activated and Ca2+-inhibited adenyl cyclase (ADCY) isoforms are expressed in insulin-secreting cells . Ca2+-activated phosphodiesterase (PDE) has been proposed to mediate cAMP oscillations in clonal MIN6 cells [14, 15]. Similarly, cAMP accumulation activates PKA, leading to phosphorylation of voltage-gated Ca2+ channels (VGCCs) on the plasma membrane [16, 17] and inositol 1,4,5-trisphosphate and ryanodine receptors on the endoplasmic membrane [18, 19]. Owing to the complexity of these interactions, the temporal and causal relationships between the Ca2+- and cAMP/PKA-mediated pathways in beta cells remain undefined [11, 13, 14, 15, 20, 21]. It is unclear whether and how glucose or depolarisation triggers activation of the cAMP/PKA pathway, and vice versa.
In the present study, we used a genetically encoded AKAR3 reporter  to monitor the real-time activity of PKA in primary beta cells using fluorescence resonance energy transfer (FRET). By combining membrane capacitance (Cm) measurements with FRET imaging in individual beta cells, we showed that depolarisation-induced Ca2+ influx activates PKA via an adenylyl cyclase 8 (ADCY8) subpool and thus helps to replenish the readily releasable pool (RRP) of insulin granules during glucose stimulation.
Detailed materials and methods are available in the electronic supplementary materials (ESM Methods).
Cell culture and transfection
Pancreatic islet beta cells from adult Wistar rats (150–250 g) were isolated and cultured as previously described . Briefly, rats were killed by cervical dislocation and islets were obtained from the pancreas by collagenase P digestion. After short-term tissue culture (4–24 h) in RPMI 1640 medium, single beta cells were isolated by treating the islets with 0.025% trypsin (Invitrogen, Carlsbad, CA, USA). Transfection was conducted using the Neon 10 μl transfection system MPK10096 (Invitrogen). Experiments were performed 24–72 h after transfection, or >72 h for ADCY8 knockdown (KD).
Real-time FRET and Ca2+ imaging
Live imaging of the AKAR3 fluorescent PKA reporter was performed using a Zeiss 710 inverted confocal microscope (Carl Zeiss, Oberkochen, Germany). AKAR3 was excited using a 405 nm laser, and a simultaneous two-channel mode was used for emission detection: one channel for cyan (466–489 nm) and the other for yellow (519–535 nm). The FRET ratio was calculated as R = FYFP/FCFP, and normalised as ΔR = ΔR′/R, where ΔR′ is the absolute change in the FRET ratio, to take into account variation in the basal FRET ratio among different cells. For simultaneous imaging of [Ca2+]i and FRET, cells were preloaded with 5 μmol/l Rhod-2 AM (Invitrogen) for <10 min, and the switched mode of frame-scan was used to alternately detect FRET and Ca2+ signals. Ca2+ signals were detected at 543 nm excitation and 560–620 nm emission. Images were acquired at 0.5 Hz, and the temperature was maintained at 28–32°C using a TempModule S (Carl Zeiss). Control solutions and drugs were applied locally to individual cells during recording using a multichannel microperfusion system .
Electrophysiology and membrane capacitance recording
Whole-cell and perforated whole-cell configurations were used as previously described [3, 25]; the latter configuration was used during imaging to avoid loss of fluorescence intensity resulting from protein leakage into the pipette. For H-89 inhibition experiments, cells were dialysed with a solution containing 15 μmol/l H-89 for >7 min. Beta cells were characterised as healthy cells with a Cm of >4 pF and no Na+ current . The standard extracellular solution contained 118 mmol/l NaCl, 20 mmol/l tetraethylammonium chloride (TEA), 5.6 mmol/l KCl, 2.6 mmol/l CaCl2·2H2O, 1.2 mmol/l MgCl2, 5 mmol/l d-glucose and 5 mmol/l HEPES at pH 7.4. The intracellular solution contained 152 mmol/l CsCH3SO3, 10 mmol/l CsCl, 10 mmol/l KCl, 1 mmol/l MgCl2 and 5 mmol/l HEPES, with pH adjusted to 7.35 using CsOH.
Detection of insulin release
Insulin release from beta cells was measured by ELISA as previously described . For ELISA, we used Krebs-Ringer buffer (KRB; 5 mmol/l KCl, 120 mmol/l NaCl, 15 mmol/l HEPES, pH 7.4, 24 mmol/l NaHCO3, 1 mmol/l MgCl2, 2 mmol/l CaCl2 and 1 mg/ml BSA). Cells were treated with 105 mmol/l KCl for 1 min and the incubation solution was then collected for analysis. After incubation for 10 min with KRB (vehicle), PKA blocker (H-89) or ADCY8 blocker (2′,5′-dideoxyadenosine [DDA]), cells were treated with KCl for a further 1 min. All samples were then centrifuged at 16,000 g for 5 min, and insulin levels in the supernatants were determined.
All data were collected and analysed using Igor software (WaveMetrix, Lake Oswego, OR, USA). The means ± SEM were calculated, and the Student’s t test was used to compare treatment effects. Statistical significance was set at p < 0.05.
Depolarisation induces PKA activation in pancreatic beta cells
Stimulation of cells with 105 mmol/l KCl for 25 s (n = 14) also evoked a robust increase in ΔR, comparable with that evoked by IBMX, followed by a slower (1–2 min) return to baseline (Fig. 1b, d). This ΔR value could not have been affected by intracellular pH because the pH did not alter during KCl treatment (ESM Fig. 1). A longer depolarisation period (100 s, n = 10) did not lead to a further increase in ΔR (Fig. 1c, d), but was associated with a longer recovery time (time constant, tau) to the basal level (tau = 171 ± 45 s). In contrast, a longer application of IBMX (n = 10) induced a much greater increase in ΔR (0.42 ± 0.05), but with a similar recovery time (tau = 54 ± 9 s) as that following a 25 s IBMX treatment (Fig. 1c, d). Therefore, membrane depolarisation-induced PKA activity is saturable and reaches its peak value after 25 s KCl stimulation.
Rather than generating persistent membrane depolarisation, as seen with KCl, glucose induces short trains of action potential spikes in beta cells [15, 26]. Therefore, we used a perforated whole-cell voltage clamp, which keeps most of the intracellular metabolites intact during recording , to measure PKA activity evoked by a single depolarisation event. A 500 ms single-step depolarisation from −70 to 0 mV, which is comparable with glucose-induced depolarisation, increased PKA activity in 25% of recorded beta cells (Fig. 1e, f; ΔR = 0.08 ± 0.01, n = 8 out of 32 recorded cells). Although the ΔR increase was smaller, the recovery rate (tau = 94 ± 19 s) after depolarisation was similar to that induced by 25 s KCl stimulation (Fig. 1e, f). Thus, physiologically relevant depolarisation conditions can activate PKA in a proportion of beta cells.
Depolarisation-induced PKA activation is Ca2+ dependent
This temporal association suggests a causal relationship between increased [Ca2+]i and PKA activation. To test this possibility, cells were sequentially stimulated with a KCl solution containing 2.6 mmol/l or 0 mmol/l Ca2+ for 25 s. When cells were depolarised in the Ca2+-free solution, there was no PKA activation (Fig. 2a, b; n = 7); however, the addition of 2.6 mmol/l Ca2+ partially restored PKA activation. Therefore, depolarisation-induced PKA activation is Ca2+ dependent.
ADCY8 is required for Ca2+-dependent PKA activation
Ca2+ modulates the intracellular free cAMP concentration ([cAMP]i) and PKA activity via either Ca2+-sensitive ADCY or Ca2+-sensitive PDE . Because depolarisation/Ca2+-induced PKA activation showed different kinetics (slower decay rate) from those evoked by PDE inhibition (Fig. 1a, b), we speculated that ADCYs may play an important role. ADCY8 is activated by Ca2+/calmodulin and participates in glucose- and GLP-1-mediated cAMP production in pancreatic beta cells [13, 28]. We therefore used RNA interference (RNAi)-based ADCY8 KD to determine the role of ADCY8 in depolarisation-evoked PKA activation.
Depolarisation-induced PKA activation replenishes depleted vesicle pools
Typically, the first depolarising pulse train led to an increase in ΔR comparable to that evoked by a 100 s KCl stimulation (Fig. 5b, e; ΔR = 0.20 ± 0.06, n = 5). The second pulse train was applied approximately 100 s later, when the cytoplasmic PKA activity had returned to the basal level (Fig. 5b). Under control conditions, Cm changes triggered by the second pulse train were similar to those triggered by the first (Fig. 5b, d; IRP2/IRP1 1.08 ± 0.07, RRP2/RRP1 0.95 ± 0.05), indicating complete replenishment of both pools (IRP and RRP). In contrast, 7–10 min after cell dialysis with a patch pipette containing H-89 (a membrane-permeable PKA inhibitor ), no increase in ΔR was induced by the first or second depolarising pulse train (Fig. 5c, e), indicating a complete blockade of PKA activation by H-89. The IRP (75 ± 12 fF) and RRP (279 ± 18 fF) induced by the first depolarising pulse train remained unchanged in the presence of H-89. However, the recovery of these pools before the second pulse train was severely impaired by PKA inhibition (Fig. 5c, d; IRP2/IRP1 0.71 ± 0.06, RRP2/RRP1 0.43 ± 0.08, n = 6).
Next, we examined insulin release from dispersed islet beta cells using ELISA. Two sequential depolarisation events 10 min apart each triggered almost the same amount of insulin release in control cells (Fig. 5f). However, cells treated with either H-89 or the transmembrane ADCY inhibitor DDA during the interval between depolarising pulse trains exhibited significantly less insulin release in response to the second vs the first stimulus (Fig. 5f; n = 4 for each experimental condition). Consistent with this, Rp-cAMPS, a more selective cAMP/PKA inhibitor, had similar inhibitory effects on both phases of glucose-induced insulin secretion (ESM Fig. 2). Taken together, these findings suggest that PKA activity stimulated by depolarisation and/or Ca2+ entry plays a central role in refilling insulin vesicle pools in rat primary beta cells.
ADCY8 activation facilitates sustained vesicle fusion
A major finding of the present study is that depolarisation-induced Ca2+ influx has dual effects on insulin release in beta cells, by directly triggering vesicle fusion (for instant release) and indirectly promoting vesicle replenishment (for sustained release). Regarding the relationship between Ca2+ and vesicle pools, it has been established that intracellular Ca2+ triggers vesicle release via stimulating Ca2+-dependent vesicle fusion to the plasma membrane in neurons and endocrine cells [24, 31, 32, 33], including beta cells [25, 34, 35, 36]. However, multiple mechanisms have been proposed to promote vesicle pool replenishment in excitable cells. In chromaffin cells and neurons, it is known that basal [Ca2+]i level can modulate the vesicle pool [37, 38]. Previous pharmacological experiments established that cAMP also facilitates replenishment of the vesicle pool in beta cells via both PKA-dependent and EPAC2-dependent pathways [2, 4, 5, 6, 34]. Here, by combining PKA imaging with Cm recording, we found that ADCY8 links the Ca2+- and cAMP/PKA-dependent pathways (described above) in rat primary beta cells. When using a physiologically relevant stimulation pattern (Fig. 3), the first depolarising pulse train caused not only the first phase of secretion and PKA activation but also facilitated exocytosis/insulin release induced by the second pulse train through vesicle pool replenishment supported by PKA activation (Figs 5 and 6). These findings suggest that Ca2+- and ADCY8-dependent PKA pathways may also play critical roles in promoting sustained insulin release upon glucose challenge under physiological conditions.
The second important finding is the causal link between depolarisation-induced Ca2+ entry and enhanced PKA activity in rat primary beta cells. Previous studies using genetic indicators of cAMP levels in clonal MIN6 cells showed coordinated glucose-stimulated oscillations in Ca2+ and cAMP [14, 20], as well as Ca2+ influx and an ADCY8-induced cAMP signal . A recent study reported that Ca2+ and cAMP oscillate in synchrony and that PKA activity lags behind cAMP changes such that an increase in PKA activity precedes the next round of Ca2+ signalling after depolarisation in MIN6 cells . Differences between stimuli (KCl/glucose vs TEA) may account for the observed differences in lag times between different signals. In the present study, we demonstrated that a physiologically relevant depolarisation-triggered transient increase in [Ca2+]i precedes the transient PKA activation by 28 s in primary beta cells (Fig. 2c). Moreover, the depolarisation-evoked PKA activation was abolished by removing extracellular Ca2+ (Fig. 2a, b). Glucose-stimulated PKA activity was also coupled to [Ca2+]i, although the lag time and the waveform linking them were variable (Fig. 3b). This variation may be caused by glucose promotion of cAMP production via metabolic coupling factors unrelated to Ca2+ [11, 20, 40]. These results established the important contribution of elevated [Ca2+]i to activation of the glucose-triggered cAMP/PKA pathway in native beta cells, as distinct from previous studies using cell lines.
Our third finding is that an ADCY8 subpool in beta cell contributes to depolarisation-induced PKA activation. ADCY8 is an important mediator of cAMP accumulation evoked by incretin stimulation . Previous studies also revealed that A kinase-anchoring protein 79/150 (AKAP79/150) specifically interacts with ADCY8 and thus has the potential to produce localised cAMP signalling evoked by local Ca2+ entry . Here, we showed that the level of PKA activation following depolarisation triggered by 105 mmol/l KCl perfusion for 25–100 s was similar to that triggered by patch-clamp depolarisation for ≤5 s (Figs 1 and 5). This was not a consequence of probe saturation because 100 s IBMX evoked an even greater response in the same cells (Fig. 1c, d). Instead, our interpretation of the data is that an ADCY8 subpool is activated by membrane depolarisation. Although ADCY8 is almost completely absent from human beta cells [12, 42], this finding may help to reveal the roles of other ADCY isoforms, such as ADCY5, which is involved in human islet function .
We thank H. Ma (Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, China), T. Xie (College of Engineering, Peking University, China) and S. Chen (College of Engineering, Peking University, China) for training in experimental techniques, I. Bruce (Institute of Molecular Medicine, Peking University, China) for his comments on the manuscript.
This work was supported by grants from the National Basic Research Programme of China (2012CB518006), the National Natural Science Foundation of China (31228010, 31171026, 31100597, 31327901, 81222020, 31221002, 31330024 and 31400708) and the National Key Technology R&D Programme (SQ2011SF11B01041). Changhe Wang was supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Science.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
HD, CW, LC, XY and ZZ were responsible for conception and design of the study. HD, CW, XW, LY, XZ, ST, HX, BL, QW, QZ, MH, YW, LW, QL, JS, YW, SS, XK and LZ contributed to data acquisition, analysis and interpretation. MR, JL and JZ contributed to data analysis and interpretation. All authors were involved in drafting the manuscript and all approved the final version. ZZ is the guarantor of this work.
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