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* This work was supported by grants from The Wellcome Trust, the Medical Research Council, and The Royal Society. The authors declare that they have no conflicts of interest with the contents of this article. † Deceased. 2 Present address: The Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, OX3 7LJ, United Kingdom.
Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells.
), like Sur1 or Kir6.2 knock-out mice), are not hypoglycemic, and islets from adult knock-out mice are still capable of responding to glucose with electrical activity, [Ca2+]i oscillations, and insulin secretion (
). The identity and regulation of this membrane conductance remain an enigma.
In contrast to the Ca2+ influx across the plasma membrane that plays a critical role in effecting insulin granule exocytosis, Ca2+ release from intracellular stores has been thought to play a modulatory rather than a triggering role in stimulus-secretion coupling in the pancreatic β cell. [Ca2+]i oscillations in response to glucose are modulated by the uptake and release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores (
). In addition, several incretins, such as glucagon-like peptide 1 and acetylcholine, are thought to enhance insulin secretion by mechanisms that are, in part, dependent on Ca2+ release from intracellular stores via intracellular messengers such as cAMP and inositol trisphosphate (IP3) (
NAADP, the most potent of the Ca2+-mobilizing messengers described, has been shown to mediate local Ca2+-signaling events by releasing Ca2+ from acidic, endolysosomal Ca2+ stores in several vertebrate and invertebrate cells (
). NAADP-induced Ca2+ release in MIN6 cells can be disrupted by the lysomotropic agent glycyl-l-phenylalanine-β-naphthylamide (GPN) or bafilomycin, which disrupts acidic store Ca2+ storage implicating lysosomally related organelles as the principal target for NAADP in these cells (
), consistent with its role as an intracellular messenger. NAADP introduced into mouse pancreatic β cells via a patch pipette was found to evoke a series of oscillatory plasma membrane currents, which were blocked by the NAADP antagonist Ned-19 (
). Furthermore, increasing concentrations of Ned-19 abolished glucose-evoked Ca2+ spiking in mouse pancreatic β cells, suggesting an important role for NAADP in stimulus-response coupling in these cells (
). At present, ADP-ribosyl cyclases, including CD38, are the only characterized enzymes that have been demonstrated to catalyze the synthesis of NAADP, using NADP and nicotinic acid as substrates by a base-exchange mechanism (
), to explore a possible role for TPC-dependent NAADP-induced Ca2+ release from acidic stores in glucose-induced [Ca2+]i increases and insulin secretion in primary mouse β cells.
This study highlights the importance of NAADP-sensitive acidic stores and the newly identified endolysosomal channels TPC1 and TPC2 in Ca2+ signaling during stimulus-secretion coupling in mouse pancreatic β cells. Since its discovery as a potent Ca2+-mobilizing agent in sea urchin egg homogenates (
In contrast to the other two principal mobilizing messengers IP3 and cADPR, the major target organelles for NAADP in sea urchin eggs are acidic stores rather than the ER. Pharmacological approaches and cell fractionation studies revealed that NAADP releases Ca2+ from a separate organelle to the ER (
). Here, we have found that Ca2+ signals evoked by the membrane-permeant NAADP analogue NAADP-AM are from intracellular stores because they persist in the absence of extracellular Ca2+ and that the NAADP antagonist Ned-19 blocks this effect (Fig. 1).
Comparison of the effects of drugs that effect Ca2+ uptake and storage in different organelles supports a role for acidic stores rather than the ER as the target of NAADP. Bafilomycin selectively inhibits vacuolar H+ pumps that acidify acidic stores, and it has been shown that Ca2+ uptake into acidic organelles is pH-dependent and probably mediated by Ca2+/H+ exchange (
). Bafilomycin treatment was thus found to abolish NAADP-AM-evoked Ca2+ release (Fig. 1C). In contrast, thapsigargin (a SERCA pump inhibitor that blocks Ca2+ uptake into the ER) was found to enhance NAADP-AM-induced Ca2+ release. This suggests that NAADP-evoked Ca2+ release in the β cell does not trigger further Ca2+ release through ER mechanisms. Rather the predominant role of the ER here is to act to buffer Ca2+ rather than as a source for release, and the functional removal of the ER decreases Ca2+ buffering, allowing Ca2+ release from acidic stores to increase further in the cytoplasm. The role of the ER to buffer Ca2+ during signaling has also been noted for glucose-evoked Ca2+ signals where glucose first decreases cytoplasmic Ca2+ due to increased ATP generation and stimulation of SERCA pumps (
). Previous studies also support acidic stores as targets for NAADP. Ca2+ indicators targeted to acidic granules or ER in MIN6 cells showed that NAADP releases Ca2+ from acidic organelles but not the ER (
). The delay in Ca2+ responses seen with NAADP-AM (FIGURE 1, FIGURE 4) may also be determined partly by the time for hydrolysis of ester groups by intracellular endogenous esterases, which varies between cells (
), uptake of Ca2+ into acidic stores might be enhanced by glucose-stimulated ATP production, and because luminal Ca2+ sensitizes TPCs to low NAADP concentrations, this could promote Ca2+ release from these stores (
) also reported that NAADP mobilizes Ca2+ from acidic stores in the INS-1 β cell line, an effect blocked by Ned-19, and evoke membrane depolarization and spike generation in this cell line. We show here that NAADP-evoked Ca2+ transients in β cells are abolished in cells prepared from Tpcn1−/− and Tpcn2−/− mice (Fig. 7, A and B). We found that endogenous TPC2 proteins in human β cells co-localized with lysosomal markers (Fig. 6B). Interestingly, TPC2 did not appear to colocalize with insulin granules, which have also been proposed to function as NAADP-sensitive Ca2+ stores in β cells (
In addition to mobilizing Ca2+ from acidic stores, NAADP was also found here to evoke plasma membrane cation currents and to depolarize the plasma membrane. NAADP applied through the patch pipette at low concentrations evoked a series of inward current transients (Fig. 3, A–G). Application of higher NAADP (100 μm) gave no response, consistent with the bell-shaped concentration-response curve for NAADP in mammalian systems and paralleling the concentration dependence of NAADP for Ca2+ release in β cells (Fig. 1A) (
). These currents were blocked by Ned-19 and by BAPTA, suggesting that they are Ca2+-activated. Their abolition by replacing Na+ with N-methyl-d-glucamine suggests that the currents are cation currents largely carried by Na+ ions. Interestingly, a nonselective cation current has also been reported to be activated by GLP-1 (where GLP-1 is glucagon-like peptide 1), an agonist that has also been reported to elevate NAADP levels (
). The identity of the channels responsible has not been established, but our results with 9-phenanthrol may tentatively point to some involvement of the TRPM4 channels. NAADP-evoked Ca2+ release has recently been shown to activate TRPM4 channels in HeLa cells (
). The NAADP-evoked currents were found to be coincident with small NAADP-evoked Ca2+ transients (Fig. 7A), and neither NAADP-evoked Ca2+ transients nor currents were observed in cells from Tpcn2−/− mice (Fig. 7B). We propose that these currents are due to NAADP-evoked Ca2+ release from endolysosomal stores via TPC2 channels and that this, in turn, via elevation of [Ca2+]i, leads to Ca2+-dependent activation of plasma membrane cation channels, possibly TRPM4 or TRPM5. Activation of these channels would then result in membrane depolarization. The finding that application of NAADP-AM elicited a series of membrane potential spikes (Fig. 4B) is consistent with this scenario and in agreement with a report that NAADP causes membrane depolarization in INS1 cells (
To investigate the role of NAADP-mediated Ca2+ signaling in glucose-induced electrical activity and [Ca2+]i, four different approaches were used to block NAADP signaling. These were as follows: (i) abrogation of Ca2+ storage by acidic stores with vacuolar proton pump inhibitors and GPN; (ii) inhibition of the NAADP receptor by Ned-19; (iii) self-desensitization of the NAADP receptor by NAADP; and (iv) knock-out of Tpcn2 and Tpcn1, genes encoding proposed NAADP target channels. Intriguingly, high glucose was also found to evoke small Ned-19-sensitive currents similar to those evoked by pipette application of NAADP (Fig. 3H). Thus, NAADP signaling may contribute, at least partly, to bringing the membrane potential from rest to the threshold for activation of VDCCs (Fig. 10). As has been recognized for a long time, closure of KATP channels is not sufficient to explain how glucose depolarizes the pancreatic β cell; a depolarizing membrane current is also required (
). We propose that NAADP/TPC1/2-dependent mobilization of Ca2+ from an acidic intracellular store results in activation of depolarizing cation-conducting plasmalemmal ion channels and that this brings the membrane potential to the threshold for action potential firing. This is consistent with our finding that in the absence of NAADP-evoked Ca2+ signals in cells from Tpcn2−/− mice, the KATP channel blocker, tolbutamide, at threshold concentrations fails to evoke Ca2+ signals as seen in wild-type cells (Fig. 8I). Indeed, it is remarkable that tolbutamide cannot by itself mimic glucose-induced Ca2+ signals but requires NAADP/TPCs. Indeed, tolbutamide will only evoke Ca2+ signals when the acidic vesicle pathway is co-stimulated either with subthreshold concentrations of NAADP/AM or with a permissive subthreshold glucose (3 mm) concentration (Fig. 8, G and H).
The final step in the stimulus-secretion coupling is the exocytosis of insulin-containing granules. In isolated islets, Ned-19 completely blocked glucose-evoked insulin secretion. Ned-19, however, had no effect on secretion evoked by depolarizing islet cells with high extracellular K+, which bypasses electrical activity and depolarizes the membrane potential to ∼−10 mV and opens the VDCCs. This finding makes it possible to discard the explanation that Ned-19 inhibits insulin secretion by an off-target effect on the exocytotic machinery. In a more in vivo setting, glucose-evoked insulin secretion from perfused whole pancreata from Tpcn2−/− and Tpcn1−/− mice was investigated. Insulin secretion stimulated by glucose (20 mm) was substantially reduced compared with that from wild-type animals (Fig. 9B). Thus, we provide evidence that NAADP signaling is an important regulator of stimulus-secretion coupling in pancreatic β cells (Fig. 10).
Surprisingly, Tpcn−/− mice are only mildly diabetic as assessed by glucose tolerance tests (Fig. 9), with a significant impairment in Tpcn1−/− mice. However, a recent study has implicated TPC2 as a novel gene for diabetic traits in mice, rats, and humans (
), with a decrease in fasting glucose and insulin levels reported in Tpcn2−/− mice. The effects of knocking out Tpcn genes in mice may result in complex phenotypes, including compensatory mechanisms, with regard to blood glucose and insulin levels because NAADP-mediated Ca2+ release has been implicated in GLUT4 translocation in murine skeletal muscle (
) have been linked to type 2 diabetes, and this could potentially be accounted for by reduced NAADP synthesis. Remarkably, in a recent study, it was found that intraperitoneal injections of NAADP could restore defective insulin secretion and blood glucose regulation in db/db mice, an animal model of type 2 diabetes (
), presumably via the NAADP transport mechanisms described above.
We propose that NAADP-evoked Ca2+ release from acidic stores via TPC2 or TPC1 channels evokes a small local Ca2+ signal that activates Ca2+-dependent cation currents in the plasma membrane. The finding that the membrane currents evoked by intracellular Ca2+ mobilization are blocked by 9-phenanthrol implicates TRPM4 channels in this process. Additional direct effects of NAADP-evoked Ca2+ release on exocytosis itself cannot be excluded at this stage, with a possible contribution from exocytotic granules themselves (
). The NAADP/TPC/acidic organelle pathway represents a new component of the glucose-evoked trigger to depolarize the plasma membrane upon KATP channel closure resulting in VDCC-mediated Ca2+ influx and insulin secretion (Fig. 10). This new pathway may offer new targets for novel diabetic therapies.
A. A. and A. G. designed the experiments, and A. A. conducted the project. A. A., J. P., G. A. R., and A. G. wrote the manuscript. M. R., L. T., K. R., and J. P. produced and characterized the Tpcn2−/− mice. F. C., T. P., and G. S. C. performed some of the [Ca2+]i measurement experiments. K. C. performed the gene expression experiments. R. P., A. M. L., and G. C. C. synthesized and characterized NAADP-AM, Ned-19, and Ned-20. A. J. M. designed experiments and produced Fig. 7. G. A. R. and E. A. B. performed immunocytochemical studies. K. S. performed insulin secretion experiments. P. J. supplied and prepared human islets. P. R., S. C. C., M. B., W. S., and Q. Z. performed secretion and cell physiological measurements. P. M. H. and P. W. T. performed the electron microscopy. All authors reviewed the results and approved the final version of the manuscript.
We gratefully acknowledge the help from the staff at the University of Oxford Biomedical Science Facility for their help in breeding and maintaining the Tpcn2−/− mice. We also thank Professor Frances Ashcroft for comments on the manuscript.
Hierarchy of the beta-cell signals controlling insulin secretion.