Glucagon-like Peptide 1 Activates Protein Kinase C through Ca2+-dependent Activation of Phospholipase C in Insulin-secreting Cells
2006; Elsevier BV; Volume: 281; Issue: 39 Linguagem: Inglês
10.1074/jbc.m604291200
ISSN1083-351X
AutoresYuko Suzuki, Hui Zhang, Naoaki Saito, Itaru Kojima, Tetsumei Urano, Hideo Mogami,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAlthough the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca2+ signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca2+ influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca2+ signaling can activate PKCα and PKCϵ at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCα and PKCϵ from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca2+-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCϵ inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCϵ-(149–164) (antp-PKCϵ) and a conventional PKC inhibitor, Gö-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCγ and 2) translocation of PKCα-DsRed and PKCϵ-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCα and PKCϵ, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose. Although the stimulatory effect of glucagon-like peptide 1 (GLP-1), a cAMP-generating agonist, on Ca2+ signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca2+ influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca2+ signaling can activate PKCα and PKCϵ at a substimulatory concentration of glucose (3 mm) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKCα and PKCϵ from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca2+-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKCϵ inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKCϵ-(149–164) (antp-PKCϵ) and a conventional PKC inhibitor, Gö-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKCγ and 2) translocation of PKCα-DsRed and PKCϵ-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKCα and PKCϵ, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose. Glucagon-like peptide 1 (GLP-1) 2The abbreviations used are: GLP-1, glucagon-like peptide 1; PKA, protein kinase A; PKC, protein kinase C; cPKC, conventional PKC; nPKC, novel PKC; DAG, diacylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; VDCC, voltage-dependent Ca2+ channel; IP3, inositol 1,4,5-trisphosphate; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich C kinase substrate; DiC8, 1,2-dioctyl-sn-glycerol; BIS, bisindolylmaleimide I; ACh, acetylcholine; 8-bromo-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; TIRFM, total internal reflection fluorescence microscopy; KRB, Krebs-Ringer buffer; 2-APB, 2-aminoethoxydiphenyl borate. is an insulinotropic peptide that is known as "incretin," a gastrointestinal hormone released from the intestinal L cell in response to a meal or an oral glucose challenge. Upon binding to its receptor, GLP-1 increases cAMP levels via G-protein-coupled activation of adenylate cyclase, leading to activation of protein kinase A (PKA) (1MacDonald P.E. El-Kholy W. Riedel M.J. Salapatek A.M. Light P.E. Wheeler M.B. Diabetes. 2002; 51: S434-S442Crossref PubMed Google Scholar, 2Gromada J. Holst J.J. Rorsman P. Pflugers Arch. 1998; 435: 583-594Crossref PubMed Scopus (221) Google Scholar). One of the mechanisms by which GLP-1 potentiates glucose-induced insulin secretion from the pancreatic β-cells is to modulate Ca2+ signaling in several ways: 1) GLP-1 induces membrane depolarization by the closure of ATP-sensitive K+ channels (KATP channels) and/or by the opening of cAMP-operated nonselective cation channels, eliciting Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) (3Holz IV, G.G. Leech C.A. Habener J.F. J. Biol. Chem. 1995; 270: 17749-17757Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar); 2) PKA-dependent phosphorylation of L-type VDCCs increases the integrated Ca2+ current by slowing the time course of inactivation and augmenting the Ca2+ current (2Gromada J. Holst J.J. Rorsman P. Pflugers Arch. 1998; 435: 583-594Crossref PubMed Scopus (221) Google Scholar, 4Suga S. Kanno T. Nakano K. Takeo T. Dobashi Y. Wakui M. Diabetes. 1997; 46: 1755-1760Crossref PubMed Google Scholar); and 3) GLP-1 promotes Ca2+ mobilization from Ca2+ stores (5Gromada J. Dissing S. Bokvist K. Renstrom E. Frokjaer-Jensen J. Wulff B.S. Rorsman P. Diabetes. 1995; 44: 767-774Crossref PubMed Scopus (119) Google Scholar). In addition to the processes mentioned above, which increase the cytosolic Ca2+ concentration ([Ca2+]i), other roles of GLP-1 as a Ca2+ modulator are gradually being elucidated. Protein kinase C (PKC) plays a role in insulin secretion that is equally important as that of cAMP/PKA signaling. Among 10 identified PKCs, conventional PKC (cPKC; PKCα, PKCβI, PKCβII, and PKCγ) is activated by Ca2+ and diacylglycerol (DAG), and novel PKC (nPKC; PKCδ, PKCϵ, PKCη, and PKCθ) is activated by DAG in a Ca2+-independent manner (6Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2367) Google Scholar, 7Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar, 8Toker A. Front. Biosci. 1998; 3: D1134-D1147Crossref PubMed Google Scholar). In general, DAG is thought to be a product of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. This hydrolysis is caused by activation of phospholipase C (PLC) following agonist binding to a G-protein-coupled receptor. We have recently demonstrated a new mechanism by which Ca2+ influx alone, via VDCCs, can generate DAG (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This occurs through Ca2+-dependent PLC activation, leading to activation of PKCα and PKCθ as representatives of cPKC and nPKC in INS-1 cells, which are an insulin-secreting cell line. An additional line of evidence shows that GLP-1 increases not only levels of cAMP but also levels of inositol 1,4,5-trisphosphate (IP3), the other product of PIP2 hydrolysis (2Gromada J. Holst J.J. Rorsman P. Pflugers Arch. 1998; 435: 583-594Crossref PubMed Scopus (221) Google Scholar, 5Gromada J. Dissing S. Bokvist K. Renstrom E. Frokjaer-Jensen J. Wulff B.S. Rorsman P. Diabetes. 1995; 44: 767-774Crossref PubMed Scopus (119) Google Scholar, 10Dillon J.S. Tanizawa Y. Wheeler M.B. Leng X.H. Ligon B.B. Rabin D.U. Yoo-Warren H. Permutt M.A. Boyd A.E. II I Endocrinology. 1993; 133: 1907-1910Crossref PubMed Scopus (104) Google Scholar). These observations prompted us to investigate whether GLP-1 can activate both cPKC and nPKC in a Ca2+-dependent manner. Recent advances in the use of fluorescent proteins, such as green fluorescent protein (GFP) and red fluorescent protein (DsRed), have allowed us to investigate PKC activity in intact living cells by monitoring translocation of GFP-tagged PKCs and related proteins (11Almholt K. Arkhammar P.O. Thastrup O. Tullin S. Biochem. J. 1999; 337: 211-218Crossref PubMed Scopus (53) Google Scholar, 12Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 13Uchino M. Sakai N. Kashiwagi K. Shirai Y. Shinohara Y. Hirose K. Iino M. Yamamura T. Saito N. J. Biol. Chem. 2004; 279: 2254-2261Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 14Zhang H. Nagasawa M. Yamada S. Mogami H. Suzuki Y. Kojima I. J. Physiol. 2004; 561: 133-147Crossref PubMed Scopus (33) Google Scholar). Using this approach, we have established the following fusion proteins as markers of PKC activity in INS-1 cells: 1) fluorescent protein-tagged PKCs, PKCα-GFP (DsRed), and PKCϵ-GFP; 2) the C1 domain of PKCγ-GFP (C12-GFP) for DAG binding as a DAG biosensor; and 3) myristoylated alanine-rich C kinase substrate (MARCKS)-GFP as a substrate of PKC. These markers enable us to probe many aspects of the mechanisms of GLP-1-evoked PKC activation using epifluorescence microscopy and total internal reflection fluorescence microscopy (TIRFM). The present study was conducted to examine whether GLP-1 activates PKCα and PKCϵ at a substimulatory concentration of glucose. Among the multiple PKC isoforms expressed in pancreatic β cells, these two proteins are likely to play a dominant role in glucose-induced insulin secretion (15Mendez C.F. Leibiger I.B. Leibiger B. Hoy M. Gromada J. Berggren P.O. Bertorello A.M. J. Biol. Chem. 2003; 278: 44753-44757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 16Yedovitzky M. Mochly-Rosen D. Johnson J.A. Gray M.O. Ron D. Abramovitch E. Cerasi E. Nesher R. J. Biol. Chem. 1997; 272: 1417-1420Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The roles of PKCα and PKCϵ in forskolin-induced insulin secretion were also evaluated in INS-1 cells and rat islets. Here we provide fresh evidence that GLP-1 can activate PKCα and PKCϵ through Ca2+-dependent activation of PLC, suggesting that GLP-1-evoked PKC activation contributes significantly to basal insulin secretion. PKCα-pEGFP and pDsRed1-N1 were obtained from Clontech (Palo Alto, CA). pEGFP of PKCα-pEGFP was replaced with pDsRed1-N1. The plasmids encoding PKCϵ-GFP, MARCKS-GFP, and C12-GFP were prepared as described previously (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 17Shirai Y. Kashiwagi K. Yagi K. Sakai N. Saito N. J. Cell Biol. 1998; 143: 511-521Crossref PubMed Scopus (123) Google Scholar). Insulin-producing INS-1 cells were a gift from Dr. Sekine (Tokyo University) (18Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar). The cells were grown in 100-mm culture dishes at 37 °C and 5% CO2 in a humidified atmosphere. The culture medium was RPMI 1640 (Sigma) with 10 mm glucose supplemented with 10% fetal bovine serum, 1 mm sodium pyruvate, 1 mm l-glutamine, and 50 μm mercaptoethanol. For fluorescence imaging, the cells were cultivated on a 35-mm glass bottom dish (Asahi Techno Glass, Japan) at 50% confluence 2 days before transfection. A plasmid encoding the GFP or DsRed-tagged proteins was transfected into the cells by lipofection using TransIT™-LT1 (Mirus, Madison, WI). Experiments were performed within 2 days of transient transfection. We established six stable transfectants from parental INS-1 cells expressing MARCKS-GFP by G418 selection and cloning. Two of these, referred to as M5 and M6, were employed for translocation experiments. The standard extracellular solution contained 140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 3 mm glucose, and 10 mm Hepes-NaOH (pH 7.3). The solution for membrane depolarization contained 105 mm NaCl, 40 mm KCl, 1 mm MgCl2, 2.5 mm CaCl2, 3 mm glucose, and 10 mm Hepes-NaOH (pH 7.3). In some experiments, CaCl2 was not included (the Ca2+-free solution contained 0.2 mm EGTA). The cells placed on a glass bottom dish were perfused continuously from a gravity-fed system. Experiments were performed in the standard extracellular solution at room temperature, unless otherwise noted. Krebs-Ringer buffer (KRB) contained 119 mm NaCl, 4.6 mm KCl, 1 mm MgSO4, 0.15 mm Na2HPO4, 0.4 mm KH2PO4, 25 mm NaHCO3, 2 mm CaCl2, 20 mm Hepes-NaOH (pH 7.3). Ionomycin, 1,2-dioctyl-sn-glycerol (DiC8), 8-bromo-cAMP, and forskolin were purchased from Sigma. Fura2-AM (hereafter termed Fura2) was from Molecular Probes, Inc. (Eugene, OR). Glucagon-like peptide 1 (human, 7–36 amide) was obtained from the Peptide Institute, Inc. (Osaka, Japan). Anti-cPKCα (H-7) and anti-nPKCϵ (C-15) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PKC inhibitors, bisindolylmaleimide I (BIS) and Gö-6976, U73122, U73433, and 2-aminoethoxydiphenyl borate (2-APB) were from Calbiochem. All other chemicals were from Sigma. A PKC-ϵ inhibitor peptide, antennapedia-PKC-(149–164) (antp-PKCϵ) (RRMKW KKERM RPRKR QGAVR RRV), was synthesized by Takara Shuzo Co., Ltd. (Tokyo, Japan). It is a tandemly synthesized peptide comprising peptides from the third α-helix of the homeodomain of antennapedia (residues 52–58, known as penetratin) (19Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 20Fischer P.M. Zhelev N.Z. Wang S. Melville J.E. Fahraeus R. Lane D.P. J. Pept. Res. 2000; 55: 163-172Crossref PubMed Scopus (143) Google Scholar) and the PKC-ϵ pseudosubstrate peptide (residues 149–164) (21Harris T.E. Persaud S.J. Jones P.M. Mol. Cell. Endocrinol. 1999; 155: 61-68Crossref PubMed Scopus (13) Google Scholar, 22Zoukhri D. Hodges R.R. Sergheraert C. Toker A. Dartt D.A. Am. J. Physiol. 1997; 272: C263-C269Crossref PubMed Google Scholar). Antennapedia (antp), used as a control, was also synthesized by Takara Shuzo. Epifluorescence Microscopy—Fluorescence images were captured at 5-s intervals using an Olympus inverted microscope (numerical aperture = 1.35, ×40, oil immersion objective) equipped with a cooled (–20 °C) coupled charge device digital camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan) and recorded and analyzed on an Aquacosmos imaging station (Hamamatsu Photonics). The excitation light source was a 150-watt xenon lamp with a high speed scanning polychromatic light source (C7773; Hamamatsu Photonics). GFP fluorescence was excited at 488 nm, whereas Fura2 for [Ca2+]i measurement was excited at wavelengths alternating between 340 and 380 nm. The emitted light was collected through a 535/45-nm band pass filter with a 505-nm dichroic mirror, and a short pass filter of 330–495 nm was used to reduce background fluorescence between the dichroic mirror and the emission filter, allowing simultaneous measurement of GFP and Fura2 fluorescence. We measured the fluorescence intensity of the GFP-tagged proteins in the cytosol of the cell, excluding the nucleus as a marker of translocation. These values (F) were normalized to each initial value (F0) so that the relative fluorescence change was referred to as F/F0. The cells transiently expressing GFP-tagged proteins were loaded with 2 μm Fura2 in the standard extracellular solution for 30 min at room temperature. We previously confirmed that we can distinguish between GFP and Ca2+ signals under this experimental condition (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The cells were washed twice and used within 2 h. The Fura2 ratio was calibrated by exposure to 10 μm ionomycin and 10 mm Ca2+ or 10 mm EGTA in the Fura2-loaded cells that were not transfected with the GFP-tagged proteins (Fmax = 4.75, Fmin = 0.47, β = 9). A dissociation constant of 150 nm for Ca2+ and Fura2 at room temperature was used (23Mogami H. Nakano K. Tepikin A.V. Petersen O.H. Cell. 1997; 88: 49-55Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). TIRFM or Evanescent Wave Microscopy—To obtain a high signal-to-noise ratio as compared with conventional epifluorescence microscopy, we installed a TIRFM unit (Olympus) into the same imaging system described above. The incidental light was introduced from the objective lens for TIRFM (Olympus; numerical aperture = 1.45, ×60) to generate the electromagnetic zone or so-called "evanescent field." The evanescent wave selectively excites fluorophores within 100 nm of the glass-water interface, which enabled us to monitor fluorescent proteins at and/or beneath the plasma membrane of a cell. GFP and DsRed were excited by a 488-nm laser, and the light emitted was collected through 535/45- and 605/50-nm emission filters, respectively. For simultaneous measurement of the relative fluorescence intensity changes of PKCα-DsRed and PKCϵ-GFP, the signals from GFP and DsRed excited by a 488-nm laser were collected simultaneously through an emission splitter (W-view; Hamamatsu Photonics) equipped with a 550-nm dichroic mirror and two emission band pass filters, 535/45 and 605/50 nm. INS-1 cells cultured on a coverslip at about 80% confluence were preincubated with KRB containing 3 mm glucose for 1–2 h and then either not treated (as a control) or treated with GLP-1 in buffer for 10 min. They were fixed with 3% paraformaldehyde in PBS for 30 min and permeabilized by 0.1% Triton X-100 for 10 min. The cells were blocked with 2% bovine serum albumin for 30 min and stained with anti-PKCα or anti-PKCϵ (1:100) for 1 h. After three washes in PBS containing 0.1% Tween 20, cells were labeled with fluorescein isothiocynate isomer 1-conjugated immunoglobulins (Dako, Carpinteria, CA) (1:50–200) for 1 h. After three washes, cells were mounted on a glass microscope slide with Dako fluorescent mounting medium and observed under an epifluorescence microscope. Male Wistar rats (200–250 g) were obtained from Imai Animal Company (Saitama, Japan). Pancreatic islets were isolated by digestion with collagenase (Wako Pure Chemical Industries, Tokyo, Japan) (24Lacy P.E. Kostianovsky M. Diabetes. 1967; 16: 35-39Crossref PubMed Scopus (2498) Google Scholar). Islets were detected by inspection under a microscope. Insulin secretion from pancreatic islets was measured in a static incubation system as described previously (25Yamada S. Komatsu M. Sato Y. Yamauchi K. Kojima I. Aizawa T. Hashizume K. Endocrinology. 2002; 143: 4203-4209Crossref PubMed Scopus (24) Google Scholar). Insulin secretion in INS-1 cells was measured using an enzyme-linked immunosorbent assay insulin kit (Seikagaku Corp., Tokyo, Japan). INS-1 cells were subcultured in 35-mm dishes and grown up to 80–90% confluence for 3–4 days. INS-1 cells and freshly isolated islets for each experimental group (5 size-matched islets/group) were preincubated in KRB buffer containing 3 mm glucose at 37 °C in a humidified incubator. The solution was then replaced with KRB alone or KRB containing various test agents. BIS and Gö-6976 were added directly with secretion solution, whereas antp-PKCϵ was added 1 h prior to the insulin secretion experiment. The stimulation time was carefully adjusted to standardize the times for solution changing and sample collection. The experiments were terminated by withdrawal of the supernatant solution after 1 h of incubation. The supernatant was then placed on an ice bath. Samples were kept at –20 °C until the insulin assay was performed. All samples were assayed in duplicate. Insulin concentration in rat islets was determined using a time-resolved immunofluorometric assay system as described previously (26Mashima H. Ohnishi H. Wakabayashi K. Mine T. Miyagawa J. Hanafusa T. Seno M. Yamada H. Kojima I. J. Clin. Invest. 1996; 97: 1647-1654Crossref PubMed Scopus (263) Google Scholar). Data are given as means ± S.E. Statistical significance was evaluated using Student's t test for paired observations. GLP-1 Triggers as Well as Amplifies Ca2+ Oscillation at a Substimulatory Concentration of Glucose—We first examined the temporal profile of the cytosolic Ca2+ concentration ([Ca2+]i) in response to GLP-1 (100 nm) in Fura2-loaded INS-1 cells. To reduce the effect of glucose on GLP-1 receptor-mediated signal transduction as much as possible, we used a standard extracellular solution containing 2.5 mm Ca2+ and 3 mm glucose, which is a substimulatory concentration for electrical activity and insulin secretion. In this condition, GLP-1 (100 nm) triggered and amplified [Ca2+]i oscillations in, respectively, more than 20 and 50% of the Fura2-loaded INS-1 cells (n = 107) (Fig. 1 and Table 1). This result is consistent with observations that GLP-1 alone can generate a Ca2+ signal at a substimulatory concentration of glucose in βTC cells and MIN6 cells (3Holz IV, G.G. Leech C.A. Habener J.F. J. Biol. Chem. 1995; 270: 17749-17757Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 27Tsuboi T. da Silva Xavier G. Holz G.G. Jouaville L.S. Thomas A.P. Rutter G.A. Biochem. J. 2003; 369: 287-299Crossref PubMed Scopus (0) Google Scholar) and that GLP-1 induces action potentials in electrically quiescent rat β cells following a 10-min exposure to a glucose-free solution (4Suga S. Kanno T. Nakano K. Takeo T. Dobashi Y. Wakui M. Diabetes. 1997; 46: 1755-1760Crossref PubMed Google Scholar). Application of 10 μm forskolin (n = 122) and 1 mm 8-bromo-cAMP (n = 150) yielded similar results (data not shown), confirming that GLP-1-initiated Ca2+ signaling occurs downstream of adenylate cyclase in INS-1 cells.TABLE 1Effect of GLP-1, forskolin, and 8-bromo-cAMP on [Ca2+]i at a substimulatory concentration of glucoseAgonistFraction of INS-1 cells responding to GLP-1, forskolin, and 8-bromo-cAMPTriggerAmplificationGLP-1 (100 nm)24 of 107 (20.5%)57 of 107 (54%)Forskolin (10 μm)21 of 122 (17.2%)80 of 122 (65.6%)Br-cAMP (1 mm)21 of 150 (14%)114 of 150 (76%) Open table in a new tab Translocation of Endogenous PKCα and PKCϵ from the Cytosol to the Plasma Membrane in Response to GLP-1—We have recently demonstrated that depolarization-evoked Ca2+ influx via VDCCs can activate cPKC and nPKC through Ca2+-dependent PLC in INS-1 cells (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). This prompted us to investigate whether GLP-1-induced Ca2+ signaling activates cPKC as well as nPKC. Thus, we examined translocation of endogenous PKCα and PKCϵ as representatives of cPKC and nPKC. Among the multiple PKC isoforms expressed in pancreatic β cells, PKCα and PKCϵ are likely to play a dominant role in glucose-induced insulin secretion (15Mendez C.F. Leibiger I.B. Leibiger B. Hoy M. Gromada J. Berggren P.O. Bertorello A.M. J. Biol. Chem. 2003; 278: 44753-44757Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 16Yedovitzky M. Mochly-Rosen D. Johnson J.A. Gray M.O. Ron D. Abramovitch E. Cerasi E. Nesher R. J. Biol. Chem. 1997; 272: 1417-1420Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 28Tian Y.M. Urquidi V. Ashcroft S.J. Mol. Cell Endocrinol. 1996; 119: 185-193Crossref PubMed Scopus (80) Google Scholar). An immunocytochemical assay clearly showed that 100 nm GLP-1 in the presence of 3 mm glucose induced translocation of endogenous PKCα and PKCϵ from the cytosol to the plasma membrane; this translocation is a marker of PKC activation (Fig. 2), indicating that GLP-1 can activate the two PKCs at a substimulatory concentration of glucose. GLP-1 Translocates MARCKS-GFP from the Plasma Membrane to the Cytosol—We next employed GFP-tagged MARCKS (MARCKS-GFP), a putative substrate for PKC (29Blackshear P.J. J. Biol. Chem. 1993; 268: 1501-1504Abstract Full Text PDF PubMed Google Scholar), as another temporal marker of PKC activation in order to substantiate our above finding of GLP-1-induced activation of PKC in a living cell. When activated PKC phosphorylates plasma membrane-anchored MARCKS, the phosphorylated form of MARCKS translocates from the plasma membrane to the cytosol (30Ohmori S. Sakai N. Shirai Y. Yamamoto H. Miyamoto E. Shimizu N. Saito N. J. Biol. Chem. 2000; 275: 26449-26457Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). This translocation is accompanied by reciprocal changes in the fluorescence intensities of MARCKS-GFP in the cytosol and at the plasma membrane (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, we measured the relative fluorescence change of MARCKS-GFP in the cytosol as a marker of translocation. Simultaneous monitoring of MARCKS translocation and [Ca2+]i in INS-1 cells transiently expressing MARCKS-GFP was performed. As seen in Fig. 3A, the application of 100 nm GLP-1 resulted in repetitive translocation of MARCKS-GFP to the cytosol following [Ca2+]i oscillations, and this translocation was synchronous with [Ca2+]i oscillations, indicating that a GLP-1-evoked Ca2+ signal can induce activation of PKC. We observed similar responses to forskolin and 8-bromo-cAMP (data not shown). PKC Inhibitors Block GLP-1-evoked MARCKS Translocation—The above observation prompted us to test whether PKC inhibitors block GLP-1-evoked translocation of MARCKS. In order to evaluate more precisely the effect of a PKC inhibitor on a GLP-1-mediated PKC signaling pathway, we established two clonal lines of INS-1 cells stably expressing MARCKS-GFP (referred to as either M5 or M6 cells), more than 80% of which responded to GLP-1. Fig. 3B shows that the amplitude of MARCKS translocation induced by GLP-1 was comparable with that elicited by either acetylcholine (ACh; 100 μm) or a depolarizing concentration of potassium (40 mm K+) (n = 28), both of which might produce sufficient DAG to activate PKC (9Mogami H. Zhang H. Suzuki Y. Urano T. Saito N. Kojima I. Petersen O.H. J. Biol. Chem. 2003; 278: 9896-9904Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). First, we tested whether a broad PKC inhibitor, BIS (1 μm), blocks GLP-1-evoked MARCKS translocation. In contrast to the data in Fig. 3B, neither GLP-1 nor ACh induced MARCKS translocation, despite the [Ca2+]i elevation in the BIS-treated M5 cells (n = 20; control n = 24) (Fig. 3C). We then plotted the GLP-1-evoked increases in the relative fluorescence intensities from MARCKS-GFP in the cytosol (dFMAR) against the peak values of [Ca2+]i in the absence or presence of BIS. Fig. 3D clearly shows that GLP-1-evoked dFMAR increased with the peak [Ca2+]i elevation, whereas there was little change in GLP-1-evoked dFMAR, irrespective of the peak [Ca2+]i elevation in the BIS-treated M5 cells. We further examined the effect of two isoform-specific PKC inhibitors, Gö-6976 (31Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), an inhibitor of conventional PKC, and antp-PKCϵ (14Zhang H. Nagasawa M. Yamada S. Mogami H. Suzuki Y. Kojima I. J. Physiol. 2004; 561: 133-147Crossref PubMed Scopus (33) Google Scholar), an inhibitor of PKCϵ, on GLP-1-evoked dFMAR. antp-PKCϵ enables the pseudosubstrate to be loaded into intact cells. These two inhibitors, Gö-6976 and antp-PKCϵ, have been previously shown to inhibit phosphorylation of MARCKS at 1 and 75 μm, respectively (14Zhang H. Nagasawa M. Yamada S. Mogami H. Suzuki Y. Kojima I. J. Physiol. 2004; 561: 133-147Crossref PubMed Scopus (33) Google Scholar). Fig. 3E shows that neither 1 μm Gö-6976 alone (n = 20; control n = 39) nor 75 μm antp-PKCϵ alone (n = 24) inhibited translocation of MARCKS induced by GLP-1. However, their combined treatment (n = 14) blocked it. These observations suggest that a GLP-1-mediated PKC signaling pathway exists. GLP-1-mediated Ca2+ Influx and Ca2+ Mobilization from Intracellular Ca2+ Stores Contribute to MARCKS Translocation—It is well established that GLP-1 elicits an increase in [Ca2+]i, which is derived from Ca2+ influx and Ca2+ mobilization from the intracellular Ca2+ store (5Gromada J. Dissing S. Bokvist K. Renstrom E. Frokjaer-Jensen J. Wulff B.S. Rorsman P. Diabetes. 1995; 44: 767-774Crossref PubMed Scopus (119) Google Scholar). We evaluated the relative contributions of these Ca2+ sources to GLP-1-mediated PKC signaling. Fig. 4A shows a representative experiment of this type (n = 45; control, n = 44). Upon removal of extracellular Ca2+, inhibition of the GLP-1-mediated Ca2+ influx led to a decrease in the two values: dFMAR and peak Ca2+ ratio (Fig. 4B). In addition to removal of the extracellular Ca2+, Ca2+ buffering in the cytosol by loading 20 μm 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (n = 18) resulted in the marked suppression of these two parameters as compared with control cells (Fig. 4B), indicating that GLP-1 induces Ca2+-signal-dependent translocation of MARCKS. We also examined the mechanism by which GLP-1 elicits Ca2+ mobilization from intracellular Ca2+ stores in the absence of extracellular Ca2+. Simultaneous monitoring of MARCKS translocation and [Ca2+]i revealed that GLP-1-evoked MARCKS translocation and [Ca2+]i elevation took place in 40% of the Fura2-loaded M5 cells examined (Fig. 4A), whereas none of these cells responded to GLP-1 in the pr
Referência(s)