Phosphatidylinositol 4-Kinase Serves As a Metabolic Sensor and Regulates Priming of Secretory Granules in Pancreatic  Cells
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Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic  cells Hervør L. Olsen*, Marianne Høy*, Wei Zhang†, Alejandro M. Bertorello‡, Krister Bokvist*§, Kirsten Capito¶, Alexander M. Efanov‡§, Bjo¨ rn Meister†, Peter Thams¶, Shao-Nian Yang‡, Patrik Rorsmanʈ, Per-Olof Berggren‡, and Jesper Gromada*§** *Islet Cell Physiology, Novo Nordisk A͞S, Novo Alle, DK-2880 Bagsvaerd, Denmark; †Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden; ‡The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden; ¶Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark; and ʈDepartment of Physiological Sciences, Lund University, BMC F11, SE-22184 Lund, Sweden Communicated by Rolf Luft, Karolinska Hospital, Stockholm, Sweden, March 5, 2003 (received for review November 15, 2002) Insulin secretion is controlled by the  cells metabolic state, and uting to the acquisition of fusion competence (6, 7). The the ability of the secretory granules to undergo exocytosis in- activities of these kinases, which lead to the sequential synthesis creases during glucose stimulation in a membrane potential-inde- of PI 4-phosphate [PI(4)P] and PI 4,5-bisphosphate [PI(4,5)P2], pendent fashion. Here, we demonstrate that exocytosis of insulin- have been proposed to account for at least part of the require- containing secretory granules depends on phosphatidylinositol ment for Mg-ATP in the priming process (6, 7). PI(4,5)P2 binds 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme specifically to the Ca2ϩ-dependent activator protein for secretion suppresses glucose-stimulated insulin secretion. Intracellular ap- (CAPS), which is required for Ca2ϩ-triggered exocytosis once plication of phosphatidylinositol 4-phosphate and phosphatidyl- ATP-dependent priming has been completed (8–10). This find- inositol 4,5-bisphosphate [PI(4,5)P2] stimulated exocytosis by pro- ing indicates that CAPS operates in parallel with the membrane moting the priming of secretory granules for release and increasing fusion machinery consisting of soluble N-ethylmaleimide- the number of granules residing in a readily releasable pool. sensitive factor attachment protein receptor (SNARE) pro- Reducing the cytoplasmic ADP concentration in a way mimicking teins (11). the effects of glucose stimulation activated PI 4-kinase and in- The pancreatic  cell represents a particularly suitable prep- creased exocytosis whereas changes of the ATP concentration in aration to study the mechanisms involved in priming because the physiological range had little effect. The PI(4,5)P2-binding changes in the metabolic state rapidly translate into increased or protein Ca2؉-dependent activator protein for secretion (CAPS) is reduced release competence of the insulin-containing secretory present in  cells, and neutralization of the protein abolished both granules. For example, a step elevation of ATP triggers exocy- 2؉ Ca - and PI(4,5)P2-induced exocytosis. We conclude that ADP- tosis within milliseconds (12), whereas ADP exerts a prompt (Ͻ5 CELL BIOLOGY induced changes in PI 4-kinase activity, via generation of PI(4,5)P2, s) inhibitory action (13). Here, we describe a mechanism by represents a metabolic sensor in the  cell by virtue of its capacity which glucose-induced reciprocal changes in the cytoplasmic to regulate the release competence of the secretory granules. ATP and ADP concentrations, via activation of PI 4-kinase, promotes PI(4,5)P2 synthesis, priming of secretory granules, and Ca2ϩ-dependent activator protein for secretion (CAPS) ͉ exocytosis ͉ refilling of RRP. These data provide a framework for the insulin ͉ phosphoinositides understanding of the amplifying action of glucose on insulin secretion that occurs independently of ATP-sensitive Kϩ- he pancreatic  cell contains 10,000–13,000 insulin- channel activity (14). Tcontaining secretory granules (1, 2). As in other neuroen- docrine cells, the secretory granules in the  cell functionally Materials and Methods belong to either a readily releasable pool (RRP) or a reserve Preparation and Culture of  Cells. Mouse pancreatic islets were pool. In  cells, the size of RRP is limited, and current estimates isolated from NMRI mice (Bomholtgaard, Denmark) as de- scribed (15). For electrophysiological experiments, the islets suggest that as few as 50–100 granules are immediately available ϩ for release (3). After the depletion of RRP, this pool is replen- were dispersed into single cells by shaking in Ca2 -free solution. ished by mobilization of new granules from the reserve pool. For subcellular fractionation studies, islets were isolated from Ultrastructural studies indicate that the number of granules ob͞ob mice (16). residing in the immediate vicinity of the plasma membrane, the docked pool, is sufficient for several hours of glucose-induced Capacitance Measurements. Exocytosis was monitored in single  insulin secretion (2). This finding has led to the proposal that cells as changes in cell membrane capacitance by using the mobilization does not require physical translocation of the standard whole-cell configuration as described (17, 18). The secretory granules and that chemical modification, priming, of pipette solution contained (in mM) 125 cesium-glutamate, 10 granules already in place may be sufficient to account for the CsCl, 10 NaCl, 1 MgCl2, 5 Hepes, 0.05 EGTA, 3 Mg-ATP, and refilling of RRP. It has been argued that release of RRP 0.01 GTP (pH 7.15 with CsOH) and 0–5 Mg-ADP. The extra- underlies first phase glucose-induced insulin secretion and that cellular medium consisted of (in mM) 118 NaCl, 20 tetraethyl- second phase release reflects the refilling of this pool (4). A detailed knowledge about distinct subsets of secretory granules 2ϩ and their respective contribution to the different phases of Abbreviations: CAPS, Ca -dependent activator protein for secretion; PI, phosphatidylino- sitol; PI(4)P, PI 4-phosphate; PI(4,5)P2, PI 4,5-bisphosphate; PAO, phenylarsine oxide; RRP, insulin secretion is desirable given that type 2 diabetes associates readily releasable pool. with abnormalities in the insulin release pattern (5). §Present address: Lilly Research Laboratories, Essener Strasse 93, D-22419 Hamburg, After docking of granules to the plasma membrane, phospha- Germany. tidylinositol (PI) 4-kinases and PI 5-kinases may act in conjunc- **To whom correspondence should be sent at the present address: Lilly Research Labora- tion to modify the two newly juxtaposed membranes, contrib- tories, Essener Strasse 93, D-22419 Hamburg, Germany. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0931282100 PNAS ͉ April 29, 2003 ͉ vol. 100 ͉ no. 9 ͉ 5187–5192 Downloaded by guest on September 25, 2021 ammonium-Cl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 5 Hepes (pH 7.40 using NaOH), and 5 D-glucose. The capacitance measurements commenced 2 min after establishment of the whole-cell config- uration to allow equilibration between the pipette solution and the cytoplasm. The stimulation protocols consisted of either individual 500-ms depolarizations or trains of fourteen 500-ms depolarizations applied at 1 Hz. In both cases, the pulses went from Ϫ70 mV to 0 mV. All phospholipids (Calbiochem) were dissolved in the pipette- filling solution by sonication for 30 min on ice. Monoclonal antibodies to PI(4)P and PI(4,5)P2 were from PerSeptive Bio- systems (Framingham, MA). Monoclonal antibody to phospha- tidylserine was obtained from ARUP Laboratories (Salt Lake City, UT). Monoclonal anti-PI 4-kinase type II-specific antibody 4C5G was from Upstate Biotechnology (Waltham, MA). Poly- clonal anti-p145 CAPS antibody was from T. F. J. Martin (University of Wisconsin, Madison). Phenylarsine oxide was from Calbiochem. All other chemicals were from Sigma. The capacitance measurements were performed at 33°C. Insulin Release Measurements. Insulin release from intact mouse pancreatic islets exposed for1hto30mMKϩ, 0.2 mM diazoxide (to clamp the membrane potential and study insulin secretion independently of ATP-sensitive Kϩ-channel activity), and 5 or 20 mM glucose were performed as outlined (19). Sucrose Density Gradient. Isolated islets (Ϸ500) from ob͞ob mice were homogenized in 300 l of buffer containing 20 mM Hepes, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and 0.5 g͞ml leupeptin, 0.5 g͞ml aprotinin, 0.5 g͞ml pepstatin, and 0.5 g͞ml antipain. Subcellular fractions were prepared as reported (20). Samples were analyzed on SDS͞PAGE by using the Laemmli buffer system. The presence of CAPS and Naϩ, Kϩ-ATPase was determined by Western blot analysis using polyclonal antibodies. Blots were developed by using ECL Fig. 1. PI(4)P and PI(4,5)P2 stimulate exocytosis in  cells. (A) Individual (Amersham Biosciences, Piscataway, NJ) as recommended by voltage-clamped mouse  cells were stimulated with single 500-ms depolar- 2ϩ the manufacturer. Protein content was determined according to izations (Vm, Top). The resultant Ca currents (ICa, Middle) and increases in cell ref. 21. Insulin content in each fraction was determined as de- capacitance (Cm, reflecting fusion of insulin-containing secretory granules scribed (22). with the plasma membrane) recorded under control conditions and in the presence of 1 M PI(4)P and 1 M PI(4,5)P2 are shown. (B) Histogram summa- Phosphoinositide Kinase Assay. Groups of 500 islets were collected rizing the capacitance increases elicited by single voltage-clamp depolariza- tions from Ϫ70 mV to 0 mV under control conditions and after inclusion of 1 in 400 l of 25 mM TES buffer (pH 6.90) containing (in mM) M of phosphatidylcholine (PC), PI, phosphatic acid (PA), PI(4)P, PI(4,5)P2,PI 5 MgCl2, 1 EGTA, 0.1 DTT, and 10 benzamidine. A homogenate 3,4-bisphosphate [PI(3,4)P2], and PI 3,4,5-trisphosphate [PI(3,4,5)P3]. (C) Ca- was prepared by sonication (10 s, 40 W) and centrifuged at pacitance increases (⌬Cm) elicited by single voltage-clamp depolarizations 100,000 ϫ g (60 min at 4°C).