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Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells*

Open AccessPublished:July 07, 2015DOI:https://doi.org/10.1074/jbc.M115.671248
      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.

      Introduction

      Pancreatic β cells are electrically excitable, and in response to elevated blood glucose concentrations, oscillatory bursts of Ca2+ action potentials mediated by VDCCs
      The abbreviations used are: VDCC
      voltage-dependent Ca2+ channel
      cADPR
      cyclic adenosine diphosphate ribose
      ER
      endoplasmic reticulum
      GPN
      glycyl-l-phenylalanine-β-naphthylamide
      IP3
      inositol 1,4,5-trisphosphate
      NAADP-AM
      nicotinic acid adenine dinucleotide phosphate acetoxymethyl ester
      TPC
      two-pore channel
      SERCA
      sarco/endoplasmic reticulum Ca2+-ATPase; cytosolic Ca2+
      NAADP
      nicotinic acid adenine dinucleotide phosphate
      BAPTA
      1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid.
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      ). In the pancreatic β cell line MIN6, and primary mouse β cells, glucose increases NAADP synthesis and hence intracellular levels (
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      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
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      ), 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 (
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      ) and were abolished in pancreatic β cells prepared from Tpcn2−/− mice (
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      NAADP mobilizes calcium from acidic organelles through two-pore channels.
      ). 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 (
      • Naylor E.
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      ). This finding is consistent with our earlier study showing that prior desensitization of NAADP-sensitive Ca2+ release mechanisms block subsequent glucose-evoked Ca2+ signals in MIN6 cells (
      • Masgrau R.
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      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ).
      Glucose (
      • Masgrau R.
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      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ) and glucagon like-peptide 1 (
      • Masgrau R.
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      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ,
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ) have both been reported to increase β cell NAADP levels, effects that may be partially dependent on the ADP-ribosyl cyclase, CD38 (
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ,
      • Shawl A.I.
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      • Higashida H.
      • Kim U.H.
      Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic beta 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 (
      • Aarhus R.
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      • Dickey D.M.
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      ,
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      ). It has been suggested that glucose stimulation increases the internalization of CD38 involving cytoskeletal changes (
      • Shawl A.I.
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      Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic beta cells.
      ) with NAADP synthetic sites associated with acidic organelles (
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ). Furthermore, glucose-evoked Ca2+ signals and insulin secretion are impaired in mouse Cd38−/− pancreatic β cells, and Cd38−/− mice show glucose intolerance (
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      CD38 disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+]i, and insulin secretion.
      ), and human CD38 autoantibodies and CD38 mutations have been shown to be associated with type-2 diabetes (
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      ). Recently, extracellular NAADP was found to be transported into mouse pancreatic β cells where it evoked Ca2+ release from acidic stores (
      • Park K.H.
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      • Shawl A.I.
      • Han M.K.
      • Lee H.C.
      • Kim U.H.
      Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic beta cells and adipocytes.
      ). Remarkably, intraperitoneal injections of NAADP were found to restore glucose-evoked insulin secretion in the db/db mouse model of type-2 diabetes and to ameliorate blood glucose regulation (
      • Park K.H.
      • Kim B.J.
      • Shawl A.I.
      • Han M.K.
      • Lee H.C.
      • Kim U.H.
      Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic beta cells and adipocytes.
      ).
      Here, we have used the cell-permeant analogue of NAADP, NAADP-AM (
      • Parkesh R.
      • Lewis A.M.
      • Aley P.K.
      • Arredouani A.
      • Rossi S.
      • Tavares R.
      • Vasudevan S.R.
      • Rosen D.
      • Galione A.
      • Dowden J.
      • Churchill G.C.
      Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signaling in intact cells.
      ), the selective cell-permeant NAADP antagonist Ned-19 (
      • Naylor E.
      • Arredouani A.
      • Vasudevan S.R.
      • Lewis A.M.
      • Parkesh R.
      • Mizote A.
      • Rosen D.
      • Thomas J.M.
      • Izumi M.
      • Ganesan A.
      • Galione A.
      • Churchill G.C.
      Discovery of a drug-like, small-molecule antagonist of the second messenger NAADP enabled by accessible virtual screening.
      ), Tpcn1−/− and Tpcn2−/− mice (
      • Calcraft P.J.
      • Ruas M.
      • Pan Z.
      • Cheng X.
      • Arredouani A.
      • Hao X.
      • Tang J.
      • Rietdorf K.
      • Teboul L.
      • Chuang K.T.
      • Lin P.
      • Xiao R.
      • Wang C.
      • Zhu Y.
      • Lin Y.
      • et al.
      NAADP mobilizes calcium from acidic organelles through two-pore channels.
      ), 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.

      Discussion

      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 (
      • Lee H.C.
      • Aarhus R.
      A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.
      ), NAADP has been widely demonstrated to evoke Ca2+ signals in an extensive range of mammalian cells, including those of both the endocrine and exocrine pancreas (
      • Zhao Y.
      • Graeff R.
      • Lee H.C.
      Roles of cADPR and NAADP in pancreatic cells.
      ). NAADP is an alternative product of multifunctional ADP-ribosyl cyclase enzymes, which is also responsible for the synthesis of the ryanodine receptor-regulating messenger cADPR (
      • Aarhus R.
      • Graeff R.M.
      • Dickey D.M.
      • Walseth T.F.
      • Lee H.C.
      ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP.
      ). Building on early studies suggesting that cADPR is an important regulator of Ca2+ signaling during secretion-coupling in pancreatic β cells (
      • Takasawa S.
      • Nata K.
      • Yonekura H.
      • Okamoto H.
      Cyclic ADP-ribose in insulin secretion from pancreatic beta cells.
      ), we now reported that NAADP also mobilizes Ca2+ in pancreatic β cells (
      • Johnson J.D.
      • Misler S.
      Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells.
      ,
      • Mitchell K.J.
      • Lai F.A.
      • Rutter G.A.
      Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta cells (MIN6).
      ,
      • Masgrau R.
      • Churchill G.C.
      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ).
      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 (
      • Genazzani A.A.
      • Galione A.
      Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca2+ from a thapsigargin-insensitive pool.
      ,
      • Lee H.C.
      • Aarhus R.
      Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose.
      ), identified as acidic lysosomally related organelles (
      • Churchill G.C.
      • Okada Y.
      • Thomas J.M.
      • Genazzani A.A.
      • Patel S.
      • Galione A.
      NAADP mobilizes Ca2+ from reserve granules, lysosome-related organelles, in sea urchin eggs.
      ). This principle was later extended to mammalian cells and NAADP release from acidic stores and has now been established in a large number cell types (
      • Morgan A.J.
      • Platt F.M.
      • Lloyd-Evans E.
      • Galione A.
      Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease.
      ,
      • Galione A.
      • Morgan A.J.
      • Arredouani A.
      • Davis L.C.
      • Rietdorf K.
      • Ruas M.
      • Parrington J.
      NAADP as an intracellular messenger regulating lysosomal calcium-release channels.
      ). In many cells, signaling domains at lysosome-ER junctions have been observed (
      • Morgan A.J.
      • Davis L.C.
      • Wagner S.K.
      • Lewis A.M.
      • Parrington J.
      • Churchill G.C.
      • Galione A.
      Bidirectional Ca2+ signaling occurs between the endoplasmic reticulum and acidic organelles.
      ). Thus, NAADP-evoked Ca2+ release from acidic stores may trigger further Ca2+ release from larger ER Ca2+ stores through the mediation of IP3 receptors and ryanodine receptors (
      • Patel S.
      • Churchill G.C.
      • Galione A.
      Coordination of Ca2+ signalling by NAADP.
      ). 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 (
      • Morgan A.J.
      • Platt F.M.
      • Lloyd-Evans E.
      • Galione A.
      Molecular mechanisms of endolysosomal Ca2+ signalling in health and disease.
      ). 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 (
      • Arredouani A.
      • Henquin J.C.
      • Gilon P.
      Contribution of the endoplasmic reticulum to the glucose-induced [Ca2+]c response in mouse pancreatic islets.
      ,
      • Gilon P.
      • Arredouani A.
      • Gailly P.
      • Gromada J.
      • Henquin J.C.
      Uptake and release of Ca2+ by the endoplasmic reticulum contribute to the oscillations of the cytosolic Ca2+ concentration triggered by Ca2+ influx in the electrically excitable pancreatic B-cell.
      ,
      • Arredouani A.
      • Guiot Y.
      • Jonas J.C.
      • Liu L.H.
      • Nenquin M.
      • Pertusa J.A.
      • Rahier J.
      • Rolland J.F.
      • Shull G.E.
      • Stevens M.
      • Wuytack F.
      • Henquin J.C.
      • Gilon P.
      SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca2+ pumps for Ca2+ homeostasis in pancreatic beta cells.
      ). 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 (
      • Mitchell K.J.
      • Lai F.A.
      • Rutter G.A.
      Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta cells (MIN6).
      ). Bafilomycin and the lysosomotropic agent GPN abolishes Ca2+ release by photolysis of caged NAADP in MIN6 cells, but it does not affect IP3-evoked Ca2+ release (
      • Yamasaki M.
      • Masgrau R.
      • Morgan A.J.
      • Churchill G.C.
      • Patel S.
      • Ashcroft S.J.
      • Galione A.
      Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells.
      ). In primary mouse β cells, NAADP-evoked Ca2+ release was inhibited by GPN, which was shown to lyse acidic stores selectively (
      • Duman J.G.
      • Chen L.
      • Palmer A.E.
      • Hille B.
      Contributions of intracellular compartments to calcium dynamics: implicating an acidic store.
      ,
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ). 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 (
      • Parkesh R.
      • Lewis A.M.
      • Aley P.K.
      • Arredouani A.
      • Rossi S.
      • Tavares R.
      • Vasudevan S.R.
      • Rosen D.
      • Galione A.
      • Dowden J.
      • Churchill G.C.
      Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signaling in intact cells.
      ), but the delay is probably largely due to initial buffering of Ca2+ by the ER because it was decreased by thapsigargin treatment. In addition, by analogy with the situation in the ER (
      • Arredouani A.
      • Guiot Y.
      • Jonas J.C.
      • Liu L.H.
      • Nenquin M.
      • Pertusa J.A.
      • Rahier J.
      • Rolland J.F.
      • Shull G.E.
      • Stevens M.
      • Wuytack F.
      • Henquin J.C.
      • Gilon P.
      SERCA3 ablation does not impair insulin secretion but suggests distinct roles of different sarcoendoplasmic reticulum Ca2+ pumps for Ca2+ homeostasis in pancreatic beta 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 (
      • Pitt S.J.
      • Funnell T.M.
      • Sitsapesan M.
      • Venturi E.
      • Rietdorf K.
      • Ruas M.
      • Ganesan A.
      • Gosain R.
      • Churchill G.C.
      • Zhu M.X.
      • Parrington J.
      • Galione A.
      • Sitsapesan R.
      TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+.
      ,
      • Rybalchenko V.
      • Ahuja M.
      • Coblentz J.
      • Churamani D.
      • Patel S.
      • Kiselyov K.
      • Muallem S.
      Membrane potential regulates nicotinic acid adenine dinucleotide phosphate (NAADP) dependence of the pH- and Ca2+-sensitive organellar two-pore channel TPC1.
      ), an effect that would be enhanced by removing competing ER stores.
      Since the initial reports linking NAADP-evoked Ca2+ release to two-pore channels (
      • Calcraft P.J.
      • Ruas M.
      • Pan Z.
      • Cheng X.
      • Arredouani A.
      • Hao X.
      • Tang J.
      • Rietdorf K.
      • Teboul L.
      • Chuang K.T.
      • Lin P.
      • Xiao R.
      • Wang C.
      • Zhu Y.
      • Lin Y.
      • et al.
      NAADP mobilizes calcium from acidic organelles through two-pore channels.
      ,
      • Zong X.
      • Schieder M.
      • Cuny H.
      • Fenske S.
      • Gruner C.
      • Rotzer K.
      • Griesbeck O.
      • Harz H.
      • Biel M.
      • Wahl-Schott C.
      The two-pore channel TPCN2 mediates NAADP-dependent Ca2+-release from lysosomal stores.
      ,
      • Brailoiu E.
      • Churamani D.
      • Cai X.
      • Schrlau M.G.
      • Brailoiu G.C.
      • Gao X.
      • Hooper R.
      • Boulware M.J.
      • Dun N.J.
      • Marchant J.S.
      • Patel S.
      Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling.
      ), there have been numerous reports of TPCs playing an essential role in mediating NAADP-evoked Ca2+ release from acidic stores (
      • Galione A.
      NAADP Receptors.
      ,
      • Patel S.
      • Ramakrishnan L.
      • Rahman T.
      • Hamdoun A.
      • Marchant J.S.
      • Taylor C.W.
      • Brailoiu E.
      The endolysosomal system as an NAADP-sensitive acidic Ca2+ store: role for the two-pore channels.
      ). However, recent evidence points to a separate NAADP-binding protein that interacts with TPCs to confer NAADP sensitivity (
      • Walseth T.F.
      • Lin-Moshier Y.
      • Jain P.
      • Ruas M.
      • Parrington J.
      • Galione A.
      • Marchant J.S.
      • Slama J.T.
      Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.
      ). Indeed, the requirement of NAADP-binding proteins may suggest that under certain circumstances these proteins may interact with multiple channel types (
      • Guse A.H.
      Linking NAADP to ion channel activity: a unifying hypothesis.
      ). This may explain a recent report of the loss of sensitivity of isolated lysosomes to NAADP (
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ,
      • Cang C.
      • Zhou Y.
      • Navarro B.
      • Seo Y.J.
      • Aranda K.
      • Shi L.
      • Battaglia-Hsu S.
      • Nissim I.
      • Clapham D.E.
      • Ren D.
      mTOR regulates lysosomal ATP-sensitive two-pore Na+ channels to adapt to metabolic state.
      ), and the finding that glucose may apparently still evoke Ca2+ signals in β cells from Tpcn1−/−/Tpcn2−/− mice (
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ), although it should be noted that the concentrations of NAADP-AM used in the report by Wang et al. (
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ) are more than 3 orders of magnitude greater than those found to be effective here. However, consistent with the data presented here, these authors (
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ) 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 (
      • Duman J.G.
      • Chen L.
      • Palmer A.E.
      • Hille B.
      Contributions of intracellular compartments to calcium dynamics: implicating an acidic store.
      ,
      • Mitchell K.J.
      • Lai F.A.
      • Rutter G.A.
      Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta cells (MIN6).
      ).
      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) (
      • Johnson J.D.
      • Misler S.
      Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells.
      ,
      • Masgrau R.
      • Churchill G.C.
      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ,
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ) and other mammalian cells (
      • Galione A.
      NAADP Receptors.
      ). 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 (
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ), in the HIT-T15 β cell line (
      • Leech C.A.
      • Habener J.F.
      Insulinotropic glucagon-like peptide-1-mediated activation of nonselective cation currents in insulinoma cells is mimicked by maitotoxin.
      ). 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 (
      • Ronco V.
      • Potenza D.M.
      • Denti F.
      • Vullo S.
      • Gagliano G.
      • Tognolina M.
      • Guerra G.
      • Pinton P.
      • Genazzani A.A.
      • Mapelli L.
      • Lim D.
      • Moccia F.
      A novel Ca2+-mediated cross-talk between endoplasmic reticulum and acidic organelles: implications for NAADP-dependent Ca2+ signalling.
      ). Moreover, TRPM4 and TRPM5 channels have been proposed to mediate in part a Ca2+-dependent depolarization of the plasma membrane in the INS-1 β cell line (
      • Cheng H.
      • Beck A.
      • Launay P.
      • Gross S.A.
      • Stokes A.J.
      • Kinet J.P.
      • Fleig A.
      • Penner R.
      TRPM4 controls insulin secretion in pancreatic beta cells.
      ,
      • Colsoul B.
      • Schraenen A.
      • Lemaire K.
      • Quintens R.
      • Van Lommel L.
      • Segal A.
      • Owsianik G.
      • Talavera K.
      • Voets T.
      • Margolskee R.F.
      • Kokrashvili Z.
      • Gilon P.
      • Nilius B.
      • Schuit F.C.
      • Vennekens R.
      Loss of high-frequency glucose-induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5−/− mice.
      ,
      • Brixel L.R.
      • Monteilh-Zoller M.K.
      • Ingenbrandt C.S.
      • Fleig A.
      • Penner R.
      • Enklaar T.
      • Zabel B.U.
      • Prawitt D.
      TRPM5 regulates glucose-stimulated insulin secretion.
      ,
      • Marigo V.
      • Courville K.
      • Hsu W.H.
      • Feng J.M.
      • Cheng H.
      TRPM4 impacts on Ca2+ signals during agonist-induced insulin secretion in pancreatic beta cells.
      ) and may play a general role as key components of membrane-based Ca2+ oscillators providing initial cell membrane depolarization for cell activation (
      • Guinamard R.
      • Demion M.
      • Launay P.
      Physiological roles of the TRPM4 channel extracted from background currents.
      ). 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 (
      • Wang X.
      • Zhang X.
      • Dong X.P.
      • Samie M.
      • Li X.
      • Cheng X.
      • Goschka A.
      • Shen D.
      • Zhou Y.
      • Harlow J.
      • Zhu M.X.
      • Clapham D.E.
      • Ren D.
      • Xu H.
      TPC proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes.
      ).
      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 (
      • Henquin J.C.
      • Nenquin M.
      • Ravier M.A.
      • Szollosi A.
      Shortcomings of current models of glucose-induced insulin secretion.
      ). 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).
      Figure thumbnail gr10
      FIGURE 10Proposed model for synergistic effects of NAADP-regulated TPC2 and KATP channels synergizes in glucose-evoked insulin secretion. NAADP-induced Ca2+ release synergizes with the KATP-dependent pathway to depolarize the plasma membrane and activate VDCCs. The ATP-mediated closure of KATP channels increases membrane resistance, which together with Ca2+-dependent depolarizing currents (possibly TRPM4/5 channels), activated by NAADP-induced Ca2+-release via TPC2 expressed on acidic stores, may depolarize the plasma membrane to threshold for VDCC activation (AS, acidic stores; NSCC, nonselective cation channel; Rm, membrane resistance; VDCC, voltage-dependent Ca2+ channels; Glc, glucose). In addition, NAADP-induced Ca2+-release together with VDCC-mediated Ca2+ influx are both required for exocytosis of insulin granules.
      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 (
      • Tsaih S.W.
      • Holl K.
      • Jia S.
      • Kaldunski M.
      • Tschannen M.
      • He H.
      • Andrae J.W.
      • Li S.H.
      • Stoddard A.
      • Wiederhold A.
      • Parrington J.
      • Ruas da Silva M.
      • Galione A.
      • Meigs J.
      • Meta-analyses of Glucose and Insulin-related Traits Consortium (MAGIC) Investigators
      • et al.
      Identification of a novel gene for diabetic traits in rats, mice, 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 (
      • Park D.R.
      • Park K.H.
      • Kim B.J.
      • Yoon C.S.
      • Kim U.H.
      Exercise ameliorates insulin resistance via Ca2+ signals distinct from those of insulin for GLUT4 translocation in skeletal muscles.
      ). Furthermore, NAADP signaling has been implicated in the action of peroxisome proliferator-activated receptor γ agonists in their insulin-sensitizing actions to ameliorate insulin resistance (
      • Song E.K.
      • Lee Y.R.
      • Kim Y.R.
      • Yeom J.H.
      • Yoo C.H.
      • Kim H.K.
      • Park H.M.
      • Kang H.S.
      • Kim J.S.
      • Kim U.H.
      • Han M.K.
      NAADP mediates insulin-stimulated glucose uptake and insulin sensitization by PPARγ in adipocytes.
      ). Future studies with tissue-specific inactivation of these genes will be required to address these questions.
      Although NAADP levels in β cells have been reported to be increased in response to elevated glucose, and also in response to the incretin hormone GLP-1 (
      • Masgrau R.
      • Churchill G.C.
      • Morgan A.J.
      • Ashcroft S.J.
      • Galione A.
      NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells.
      ,
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ), the mechanisms are not well understood. However, ADP-ribosyl cyclases have been implicated in NAADP synthesis in β cells (
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ). Although CD38 is a membrane-bound ecto-enzyme, glucose treatment of β cells induces endocytosis of CD38 that requires cytoskeletal changes (
      • Shawl A.I.
      • Park K.H.
      • Kim B.J.
      • Higashida C.
      • Higashida H.
      • Kim U.H.
      Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic beta cells.
      ). Inhibition of CD38 internalization with jasplakinolide, which promotes actin polymerization, blocks glucose-stimulated NAADP levels and impairs glucose-evoked Ca2+ signaling (
      • Shawl A.I.
      • Park K.H.
      • Kim B.J.
      • Higashida C.
      • Higashida H.
      • Kim U.H.
      Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic beta cells.
      ). NAADP synthesis is found to be associated with lysosomal membrane fractions (
      • Kim B.J.
      • Park K.H.
      • Yim C.Y.
      • Takasawa S.
      • Okamoto H.
      • Im M.J.
      • Kim U.H.
      Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that is essential for insulin secretion in mouse pancreatic islets.
      ). We have previously argued that cADPR (and NAADP) synthesis may occur within the acidic organelles (
      • Davis L.C.
      • Morgan A.J.
      • Ruas M.
      • Wong J.L.
      • Graeff R.M.
      • Poustka A.J.
      • Lee H.C.
      • Wessel G.M.
      • Parrington J.
      • Galione A.
      Ca2+ signaling occurs via second messenger release from intraorganelle synthesis sites.
      ). The luminal acidic pH of acidic organelles would provide an optimal environment for NAADP synthesis by ADP-ribosyl cyclases (
      • Aarhus R.
      • Graeff R.M.
      • Dickey D.M.
      • Walseth T.F.
      • Lee H.C.
      ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP.
      ). We showed that pyridine nucleotides are transported into organelles, and second messenger products are transported into the cytoplasm to their site of action (
      • Davis L.C.
      • Morgan A.J.
      • Ruas M.
      • Wong J.L.
      • Graeff R.M.
      • Poustka A.J.
      • Lee H.C.
      • Wessel G.M.
      • Parrington J.
      • Galione A.
      Ca2+ signaling occurs via second messenger release from intraorganelle synthesis sites.
      ).
      Antibodies to CD38 (
      • Pupilli C.
      • Giannini S.
      • Marchetti P.
      • Lupi R.
      • Antonelli A.
      • Malavasi F.
      • Takasawa S.
      • Okamoto H.
      • Ferrannini E.
      Autoantibodies to CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in Caucasian patients with diabetes: effects on insulin release from human islets.
      ) and a missense mutation in the CD38 gene (
      • Yagui K.
      • Shimada F.
      • Mimura M.
      • Hashimoto N.
      • Suzuki Y.
      • Tokuyama Y.
      • Nata K.
      • Tohgo A.
      • Ikehata F.
      • Takasawa S.
      • Okamoto H.
      • Makino H.
      • Saito Y.
      • Kanatsuka A.
      A missense mutation in the CD38 gene, a novel factor for insulin secretion: association with type II diabetes mellitus in Japanese subjects and evidence of abnormal function when expressed in vitro.
      ) 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 (
      • Park K.H.
      • Kim B.J.
      • Shawl A.I.
      • Han M.K.
      • Lee H.C.
      • Kim U.H.
      Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic beta cells and adipocytes.
      ), presumably via the NAADP transport mechanisms described above.

      Conclusions

      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 (
      • Mitchell K.J.
      • Lai F.A.
      • Rutter G.A.
      Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta cells (MIN6).
      ,
      • Davis L.C.
      • Morgan A.J.
      • Chen J.L.
      • Snead C.M.
      • Bloor-Young D.
      • Shenderov E.
      • Stanton-Humphreys M.N.
      • Conway S.J.
      • Churchill G.C.
      • Parrington J.
      • Cerundolo V.
      • Galione A.
      NAADP activates two-pore channels on T cell cytolytic granules to stimulate exocytosis and killing.
      ). 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.

      Author Contributions

      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.

      Acknowledgments

      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.

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