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J. Biol. Chem., Vol. 281, Issue 39, 28499-28507, September 29, 2006

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Glucagon-like Peptide 1 Activates Protein Kinase C through Ca²⁺-dependent Activation of Phospholipase C in Insulin-secreting Cells^*

Yuko Suzuki, Hui Zhang, Naoaki Saito^¶, Itaru Kojima, Tetsumei Urano, and Hideo Mogami¹

From the Department of Physiology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan, the Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan, and the ^¶Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657-8501, Japan

Received for publication, May 4, 2006 , and in revised form, July 21, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 PKC and PKC at a substimulatory concentration of glucose (3 mM) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKC and PKC 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 PKC inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKC-(149–164) (antp-PKC) and a conventional PKC inhibitor, Gö-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122 [GenBank] , 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 PKC and 2) translocation of PKC-DsRed and PKC-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKC and PKC, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Glucagon-like peptide 1 (GLP-1)² 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) (1, 2). One of the mechanisms by which GLP-1 potentiates glucose-induced insulin secretion from the pancreatic -cells is to modulate Ca²⁺ signaling in several ways: 1) GLP-1 induces membrane depolarization by the closure of ATP-sensitive K⁺ channels (K_ATP channels) and/or by the opening of cAMP-operated nonselective cation channels, eliciting Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) (3); 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 (2, 4); and 3) GLP-1 promotes Ca²⁺ mobilization from Ca²⁺ stores (5). 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; PKC, PKCI, PKCII, and PKC) is activated by Ca²⁺ and diacylglycerol (DAG), and novel PKC (nPKC; PKC, PKC, PKC, and PKC) is activated by DAG in a Ca²⁺-independent manner (6–8). 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 (9). This occurs through Ca²⁺-dependent PLC activation, leading to activation of PKC and PKC 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 (2, 5, 10). 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 (11–14). Using this approach, we have established the following fusion proteins as markers of PKC activity in INS-1 cells: 1) fluorescent protein-tagged PKCs, PKC-GFP (DsRed), and PKC-GFP; 2) the C1 domain of PKC-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 PKC and PKC at a substimulatory concentration of glucose. Among the multiple PKC isoforms expressed in pancreatic cells, these two proteins are likely to play a dominant role in glucose-induced insulin secretion (15, 16). The roles of PKC and PKC 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 PKC and PKC through Ca²⁺-dependent activation of PLC, suggesting that GLP-1-evoked PKC activation contributes significantly to basal insulin secretion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmid Construction
PKC-pEGFP and pDsRed1-N1 were obtained from Clontech (Palo Alto, CA). pEGFP of PKC-pEGFP was replaced with pDsRed1-N1. The plasmids encoding PKC-GFP, MARCKS-GFP, and C1₂-GFP were prepared as described previously (9, 17).

Cell Culture and Transfection
Insulin-producing INS-1 cells were a gift from Dr. Sekine (Tokyo University) (18). 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^TM-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.

Solutions
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).

Materials
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-cPKC (H-7) and anti-nPKC (C-15) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). PKC inhibitors, bisindolylmaleimide I (BIS) and Gö-6976, U73122 [GenBank] , U73433 [GenBank] , and 2-aminoethoxydiphenyl borate (2-APB) were from Calbiochem. All other chemicals were from Sigma. A PKC- inhibitor peptide, antennapedia-PKC-(149–164) (antp-PKC) (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 -helix of the homeodomain of antennapedia (residues 52–58, known as penetratin) (19, 20) and the PKC- pseudosubstrate peptide (residues 149–164) (21, 22). Antennapedia (antp), used as a control, was also synthesized by Takara Shuzo.

Imaging Experiments
Epifluorescence Microscopy—Fluorescence images were captured at 5-s intervals using an Olympus inverted microscope (numerical aperture = 1.35, x40, 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 (9). 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 (F_max = 4.75, F_min = 0.47, = 9). A dissociation constant of 150 nM for Ca²⁺ and Fura2 at room temperature was used (23).

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, x60) 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 PKC-DsRed and PKC-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.

Immunocytochemistry
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-PKC or anti-PKC (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.

Measurement of Insulin Secretion
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) (24). Islets were detected by inspection under a microscope. Insulin secretion from pancreatic islets was measured in a static incubation system as described previously (25). 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-PKC 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 (26).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. GLP-1 triggers as well as amplifies Ca2+ oscillation at a substimulatory concentration of glucose in INS-1 cells. [Ca2+]i measurement in the Fura2-loaded INS-1 cells was performed at 3 mM glucose. Representative traces are shown. GLP-1 (100 nM) triggered (top) and amplified (bottom) Ca2+ oscillations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 TC cells and MIN6 cells (3, 27) and that GLP-1 induces action potentials in electrically quiescent rat cells following a 10-min exposure to a glucose-free solution (4). 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.

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 (29), 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 (30). This translocation is accompanied by reciprocal changes in the fluorescence intensities of MARCKS-GFP in the cytosol and at the plasma membrane (9). 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 (9). 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 (dF_MAR) against the peak values of [Ca²⁺]_i in the absence or presence of BIS. Fig. 3D clearly shows that GLP-1-evoked dF_MAR increased with the peak [Ca²⁺]_i elevation, whereas there was little change in GLP-1-evoked dF_MAR, 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 (31), an inhibitor of conventional PKC, and antp-PKC (14), an inhibitor of PKC, on GLP-1-evoked dF_MAR. antp-PKC enables the pseudosubstrate to be loaded into intact cells. These two inhibitors, Gö-6976 and antp-PKC, have been previously shown to inhibit phosphorylation of MARCKS at 1 and 75 µM, respectively (14). Fig. 3E shows that neither 1 µM Gö-6976 alone (n = 20; control n = 39) nor 75 µM antp-PKC 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.

View larger version (124K): [in this window] [in a new window]   FIGURE 2. GLP-1 induces translocation of endogenous PKC and PKC from the cytosol to the plasma membrane in INS-1 cells. This immunocytochemical study was performed using antibodies against PKC and PKC with stimulation of GLP-1 (100 nM) for 10 min in KRB containing 3 mM glucose. The distribution of PKC and PKC was observed under control conditions (left panel) and after GLP-1 stimulation (right panel). GLP-1-induced translocation of the two PKCs (PKC (upper panel) and PKC (lower panel)) to the plasma membrane is shown. Scale bar, 10 µm. Representative images are shown from six independent experiments.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. GLP-1 translocates MARCKS-GFP from the plasma membrane to the cytosol, and GLP-1-evoked translocation of MARCKS is inhibited by PKC inhibitors. Simultaneous monitoring of MARCKS (closed circles) translocation as a marker of PKC activation and [Ca2+]i (open circles) is shown. Images were taken at the times indicated by the arrows in each image. The region of interest in the cytosol is indicated (white boxes). When MARCKS-GFP was translocated from the plasma membrane to the cytosol, the relative fluorescence of MARCKS-GFP in the cytosol increased. Scale bar, 10 µm. A, GLP-1-induced MARCKS translocation was synchronous with [Ca2+]i in Fura2-loaded INS-1 cells transiently expressing MARCKS-GFP (n = 15). B, relative change in the fluorescence intensity of MARCKS-GFP in response to GLP-1 (100 nM), ACh (100 µM), and 40 mM K+ in stable transfectants expressing MARCKS-GFP (M5) (n = 28, seven independent experiments). C, M5 cells were preincubated with Fura2 and 1 µM BIS, a broad PKC inhibitor. GLP-1-evoked MARCKS translocation was greatly suppressed despite the increase in [Ca2+]i in the presence of 1 µM BIS. D and E, scattered plots of the GLP-1-evoked increase in the relative fluorescence intensity of MARCKS-GFP (dFMAR) in the cytosol versus the peak value of [Ca2+]i. D, effect of BIS on GLP-1-induced MARCKS translocation. BIS caused little change in MARCKS translocation despite the increase in [Ca2+]i. Open circle, control cells (n = 24); closed triangle, cells treated with 1 µM BIS (n = 20; five independent experiments). E, effect of isoform-specific PKC inhibitors, antp-PKC and Gö-6976, on GLP-1-induced MARCKS translocation. Open diamond, control (n = 39); open triangle, 75µM antp-PKC (n = 24); asterisk, 1µM Gö-6976 (n = 20); closed rectangle, 75 µM antp-PKC and 1 µM Gö-6976 (n = 14). Alone, neither antp-PKC nor Gö-6976 inhibited GLP-1-evoked MARCKS translocation, but together the two inhibitors resulted in great suppression of MARCKS translocation.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. GLP-1-mediated MARCKS translocation is Ca2+-dependent. A, GLP-1-induced translocation of MARCKS-GFP (closed circles) from the plasma membrane to the cytosol coupled with an increase in [Ca2+]i (open circles) in Fura2-loaded M5 cells (n = 18) in extracellular Ca2+-free solution containing 0.2 mM EGTA. B, scattered plots of the GLP-1-evoked increase in the relative fluorescence intensity of MARCKS-GFP (dFMAR) in the cytosol versus the peak value of [Ca2+]i. M5 cells were loaded with 20 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester and 2 µM Fura2 for 30 min. The combination of Ca2+ buffering in the cytosol using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester and the removal of extracellular Ca2+ resulted in almost complete suppression of MARCKS translocation, indicating that GLP-1 translocates MARCKS in a Ca2+-dependent manner. Open circle, control (n = 44); asterisk, EGTA (n = 45); closed diamond, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (n = 18).

View larger version (12K): [in this window] [in a new window]   FIGURE 5. The putative PLC inhibitor U73122 inhibits GLP-1-evoked MARCKS translocation. Shown are scattered plots of the increase in the relative fluorescence intensity of MARCKS-GFP (dFMAR) in the cytosol evoked by 10 µM forskolin versus the peak value of [Ca2+]i. The stable transfectants expressing MARCKS-GFP(M6) cells were preincubated with either U73122 (7.5 µM) or its inactive analog U73433 (7.5 µM) and Fura2 for 40 min. MARCKS translocation was greatly inhibited in M6 cells pretreated with U73122 but not with U73433. Open circle, U73433 (n = 21); closed triangle, U73122 (n = 21).

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Temporal profile of PKC and PKC translocation in response to forskolin in the presence or absence of Ca2+ using TIRFM. PKC-DsRed and PKC-GFP were co-transfected into INS-1 cells. A and B, representative data (n = 8) for the relative change in fluorescence intensity (F/F0) of PKC-DsRed (closed circles) and PKC-GFP (open circles) at the plasma membrane, showing their translocation in response to forskolin in the presence of Ca2+. A, the entire time course of translocation of the two PKCs in response to forskolin, ACh, and 40 mM K+. Translocation of PKC induced by ACh was comparable with that in response to 40 mM K+, whereas the F/F0 of PKC in response to 40 mM K+ was far larger than that induced by ACh. B, expansion of the area outlined by the dashed box in A. Repetitive translocation of the two PKCs took place in response to 10 µM forskolin. C, forskolin- and ACh-evoked translocation of the two PKCs in Ca2+-free extracellular solution containing 0.2 mM EGTA. Representative data (n = 10) show that transient translocation of PKC, but not PKC, was induced by forskolin.

View larger version (42K): [in this window] [in a new window]   FIGURE 7. Single-cell measurement of the forskolin-induced increase in DAG content by TIRFM. C12-GFP-expressing cells were first treated with forskolin and then perfused with nominally Ca2+-free extracellular solution followed by consecutive applications of DiC8 (metabolizable DAG analogue) at 1, 3, and 10 µM. a–e were taken at the times indicated by the arrows in the top. The region of interest represented by the dashed box was at the plasma membrane. The forskolin-evoked increase in DAG concentration was about 1.44 ± 0.48 µM, as calculated from the calibration curves that provided the relative fluorescence intensities (n = 20, mean ± S.E.).

Effect of PKC Inhibitors on Forskolin-stimulated Insulin Secretion in INS-1 Cells and Rat Islets—We examined the effect of 1 µM BIS in INS-1 cells and of Gö-6976 and antp-PKC in rat islets on forskolin-stimulated insulin secretion at a substimulatory concentration of glucose (3 mM). At a concentration of 1 µM, BIS blocked MARCKS translocation irrespective of an increase in [Ca²⁺]_i (Fig. 3, C and D). As shown in Fig. 8A, forskolin-stimulated insulin secretion was significantly inhibited in BIS-treated cells as compared with control cells. We further tested the isoform-specific roles of the two PKCs in forskolin-stimulated insulin secretion in rat islets by using Gö-6976 and antp-PKC. These inhibitors did not significantly affect insulin secretion per se in rat islets at 3 mM glucose in our previous report (see Figs. 4E and 5, A and C, in Ref. 14). Forskolin-induced insulin secretion at 3 mM glucose was significantly reduced by Gö-6976 and was markedly reduced by 75 µM antp-PKC (Fig. 8B) These results indicate that a PKC signaling pathway mediated by a cAMP-generating agonist plays an important role in insulin secretion at a substimulatory concentration of glucose.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. PKC inhibitors inhibit forskolin-induced insulin secretion. A, INS-1 cells were incubated for 1 h with KRB containing 3 mM glucose in the absence (open column) or presence (closed column) of BIS. Data are mean ± S.E. of four independent experiments. B, freshly isolated islets were incubated at 3 mM glucose for 60 min with stimulation of forskolin in the presence of 1 µM Gö-6976, 75 µM antp-PKC, or 75 µM antennapedia. Data are mean ± S.E. of 10 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GLP-1-mediated Activation Mechanism of the PKC Signaling Pathway through Ca²⁺-dependent PLC Activation—We have demonstrated in this study 1) that GLP-1-induced translocation of MARCKS was inhibited by a variety of PKC inhibitors (Fig. 3, C and D) and 2) blocked by Ca²⁺ buffering (Fig. 4B); 3) that U73122 [GenBank] blocked forskolin-evoked translocation of MARCKS; 4) that application of GLP-1 and forskolin resulted in translocation of PKC and PKC (Fig. 2 and Fig. 6); and 5) that forskolin induced an increase in DAG. We also demonstrated in our previous report that Ca²⁺ influx via VDCCs can activate PLC (9). The simplest explanation for these collective observations is that cAMP-generating agonists activate PLC in aCa²⁺-dependent manner, thereby leading to activation of the two PKCs. These observations also show that Ca²⁺ signaling is essential for GLP-1-evoked PKC activation. Although we do not know definitively at present whether GLP-1-mediated PKC signaling pathways require either a Ca²⁺ signal alone or a combined signal from Ca²⁺ and cAMP, the following mechanism may be envisaged. IP₃ receptor sensitization induced by PKA phosphorylation triggers IP₃-induced Ca²⁺ release (27), which in turn may activate a Ca²⁺-dependent PLC pathway. This is consistent with the two observations that 2-APB inhibited Ca²⁺ release induced by GLP-1 upon removal of the extracellular Ca²⁺ and that Ca²⁺ release alone can activate PKC (Fig. 6C). Two PLC isoforms, PLC and PLC, may be responsible for GLP-1-mediated PKC signaling pathways. Among all known PLCs, PLC isoforms have the highest sensitivity to Ca²⁺ (38), so that a GLP-1-evoked Ca²⁺ signal could be sufficient to trigger activation of PLC. Recent studies have shown that the action of cAMP on insulin secretion is mediated not only by PKA but also by cAMP-binding proteins designated as cAMP-regulated guanine nucleotide exchange factors (cAMP-GEF or Epac) (39). cAMP activates Epac1, which catalyzes activation of GTP loading on Rap 2B, leading to PLC activation (40). Thus, PLC may also be a potential candidate for GLP-1-mediated PKC signaling pathways. (However, in opposition to this hypothesis and our observations is a report that no inositol phosphate accumulation is detected in INS-1 cells after stimulation using either GLP-1 (100 nM) or an Epac-selective cAMP analog, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (up to 300 µM) (41). We cannot clearly explain this discrepancy. It might be that we have shown that a cAMP-mediated PKC signaling pathway exists by using a single-cell-based analysis, whereas the other study measured inositol phosphate accumulation by a whole-cell population-based analysis using ³H-labeled inositol, which might not be sensitive enough to detect subtle changes in inositol phosphate content.

The Contribution of the PKC Signaling Pathway to Forskolin-stimulated Insulin Secretion Is Substantial at a Substimulatory Concentration of Glucose—GLP-1 is known to play a dual role in cell function: 1) it stimulates insulin secretion per se at a substimulatory concentration of glucose (27), and 2) it renders cells glucose-competent (42). We confirmed the first role of GLP-1 in this study. In addition, we have shown here that forskolin-induced insulin secretion is mediated partly by PKC signaling pathways (Fig. 8A). These results suggest that the relative contribution of GLP-1-mediated PKC signaling to GLP-1-induced insulin secretion is substantial at a substimulatory concentration of glucose. PKC played a dominant role over PKC in forskolin-induced insulin secretion of the rat islets (Fig. 8B). This result was reconciled with evidence of the dominant translocation of PKC induced by GLP-1 irrespective of extracellular Ca²⁺ (Fig. 6, B and C). Several isoforms of PKC are expressed in insulin-secreting cells as well as the rat pancreatic islet (16, 28). When we monitored MARCKS translocation as a marker of PKC activation, BIS and U73122 [GenBank] blocked it, but neither Gö-6976 nor antp-PKC did (Figs. 3, D and E, and 5). This is probably because of functional redundancy between the PKC isoforms. Among the PKC isoforms, however, PKC and PKC might be responsible for an agonist-stimulated insulin secretion (Fig. 8) (15, 16). The stimulatory role of GLP-1 on insulin secretion at a substimulatory concentration of glucose is very important, especially when one considers the effect of insulin secretion during a fasting period. GLP-1 is actually secreted not only during the postprandial period but also during fasting periods (43); therefore, it must be involved in basal insulin secretion mechanisms. Excessive secretion of GLP-1 might cause reactive hypoglycemia, as seen in dumping syndrome (43).

Conclusion—In this study, we have shown that GLP-1, a cAMP-generating agonist, activates conventional PKC and novel PKC through Ca²⁺-dependent PLC-mediated activation of INS-1 cells; in addition, we have measured the amount of DAG evoked by a cAMP-generating agonist in single living cells. In conclusion, GLP-1 may play a pivotal role in basal insulin secretion at substimulatory concentrations of glucose through the ternary signaling of a cAMP/PKA-Ca²⁺-PKC pathway, and these signals cannot be segregated.

	FOOTNOTES

 
^* This work was supported by a grant-in-aid for scientific research from the Ministry of Science, Education, Sports, and Culture of Japan and grants from the Takeda Science Foundation and Toukai Foundation for Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. Tel.: 81-53-435-2249; Fax: 81-53-435-7020; E-mail: hmogami{at}hama-med.ac.jp.

² The 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.

³ Y. Suzuki and H. Mogami, unpublished data.

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