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Oleic acid interacts with GPR40 to induce Ca²⁺ signaling in rat islet -cells: mediation by PLC and L-type Ca²⁺ channel and link to insulin release

Department of Physiology, Division of Integrative Physiology, Jichi Medical School, Minamikawachi, Tochigi, Japan

Submitted 27 January 2005 ; accepted in final form 11 May 2005

	ABSTRACT

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It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic -cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in -cells are little known. The present study was aimed at elucidating GPR40-linked Ca²⁺ signaling mechanisms in rat pancreatic -cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single -cells by fura 2 fluorescence imaging. OA at 1–10 µM concentration-dependently increased [Ca²⁺]_i in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca²⁺]_i increases at 11.2 mM glucose were inhibited in -cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca²⁺]_i increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 [GenBank] and neomycin, Ca²⁺-free conditions, and an L-type Ca²⁺ channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca²⁺ channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca²⁺]_i via PLC- and L-type Ca²⁺ channel-mediated pathway in rat islet -cells, which may be link to insulin release.

G protein-coupled receptor; intracellular calcium; insulin secretion; free fatty acids; phospholipase C

The putative physiological relevance is that elevation of circulating levels of FFAs upon fasting may assist an efficient insulin release when plasma glucose concentration increases in response to meals (8, 25). The effects of FFAs are thought to be due to their uptake into cytoplasm, followed by their metabolism and conversion to long-chain fatty acyl-CoA (LC-CoA; see review in Ref. 4). Exogenous FFAs elevate LC-CoA in clonal -cell lines (21, 29). An increase in cytosolic LC-CoA activates or modulates various processes in -cells: activation of protein kinase C (PKC) (1, 35), modulation of ATP-sensitive potassium (K_ATP) channel activity (18), stimulation of Ca²⁺-ATPases (7), and insulin exocytosis (6).

Recently, three groups independently reported that FFAs are ligands for GPR40, an orphan GTP-binding protein (G protein)-coupled receptor (3, 15, 16). In the rat, GPR40 mRNA is highly expressed in the pancreas, in which it is detected mainly in islet -cells by in situ hybridization (15). A significant amount of GPR40 mRNA is also detected in -cell lines (3, 15). Moreover, it was reported that FFAs activate GPR40 to amplify glucose-stimulated insulin secretion from rat pancreatic islets and an insulinoma -cell line, MIN6 cells (15). However, postreceptor mechanisms underlying GPR40-mediated insulin secretion are little known. The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) plays a central role in insulin secretion. In the present study, we aimed to determine whether FFAs act on GPR40 to increase [Ca²⁺]_i in normal rat islet -cells and, if so, to explore the mechanism that links the receptor stimulation to [Ca²⁺]_i signaling. We employed oleic acid (OA), because this FFA exists abundantly in plasma of rats and humans and shows the highest affinity for the rat GPR40 among physiologically occurring FFAs (15). OA is a monounsaturated fatty acid with a chain consisting of 18 carbon atoms. We found that OA interacts with GPR40 to increase [Ca²⁺]_i via phospholipase C (PLC) and L-type Ca²⁺ channel-dependent pathways in rat pancreatic -cells, which may be linked to insulin release.

	MATERIALS AND METHODS

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Solutions and chemicals. Measurements were carried out in HEPES-Krebs-Ringer bicarbonate buffer (KRBH) composed of (in mM) 129 NaCl, 5.0 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 2.0 CaCl₂, 1.2 MgSO₄, and 10 HEPES at pH 7.4 supplemented with 0.01% bovine serum albumin fraction V (Roch, Penzberg, Germany). Fetal bovine serum (FBS) was from Equitec-Bio (Kerrville, TX). Collagenase type XI, thapsigargin, and tolbutamide were from Sigma (St. Louis, MO). Fura 2-acetoxymethyl ester and fura 2-free acid were from Dojin Chemical (Kumamoto, Japan). Calcium Calibration Buffer kit no. 2 was from Molecular Probes (Leiden, The Netherlands). Silencer Negative Control no. 1 small interfering (si)RNA was from Ambion (Austin, TX). qPCR Mastermix Plus for SYBR Green I was from Eurogentec (Aeraing, Belgium). Minimum essential medium (MEM), Lipofectamin 2000, TRIzol reagent, and Superscript II were from Invitrogen (Carlsbad, CA). All other chemicals were from Wako Pure Chemicals (Osaka, Japan).

Isolation and culture of single islet cells. Wistar rats aged 10–12 wk (SLC, Hamamatsu, Japan) were deeply anesthetized with an intraperitoneal injection of 25% carbamic acid ethyl ester (5 ml/kg body wt). Isolation of islets and their dispersion into single islet cells were carried out as previously described (31, 32). Briefly, 1 mg/ml collagenase type XI dissolved in 5 mM Ca²⁺-containing KRBH was injected into the common bile duct at the distal end. The pancreas was removed and incubated at 37°C for 16 min. Islets were hand-collected under a microscope and then dispersed into single cells by treatment with Ca²⁺-free KRBH containing 1 mM EGTA. The single cells were plated on coverslips and maintained in culture for 1 day in MEM containing 5.6 mM glucose, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% FBS at 37°C in 95% air with 5% CO₂.

Measurements of [Ca²⁺]_i in -cells. Cytosolic [Ca²⁺]_i was measured by dual-wavelength fura 2 micofluorometry and digital imaging as previously reported (31, 32). Single cells on coverslips were incubated with 1 µM fura 2-acetoxymethyl ester for 30 min at 37°C in KRBH containing 2.8 mM glucose. Cells were then mounted in a chamber and superfused with KRBH at a rate of 1 ml/min at 37°C. The fura 2-loaded cells were excited at 340 and 380 nm alternately, the emission signals at 510 nm were detected every 5 or 10 s by a cooled charge-coupled device camera, and the ratio was produced by an AquaCosmos system (Hamamatsu Photonics, Hamamatsu, Japan). Ratio values were converted to [Ca²⁺]_i according to calibration curves obtained from the relationship between free Ca²⁺ concentration and the ratio determined in a cytosol-mimicking solution using a calcium calibration buffer kit and fura 2-free acid.

[Ca²⁺]_i data were obtained only from the -cells that were selected by immunostaining with anti-insulin antiserum or by morphological and physiological criteria for these -cells reported previously (33, 34). When increases in [Ca²⁺]_i took place within 3 min after addition of agents and their amplitudes were 50 nM or larger, they were considered responses.

Immunocytochemistry. The cells were fixed with 10% formalin in phosphate-buffered saline (PBS) for 30 min. They were washed with PBS, and then treated with 1% normal horse serum and 0.4% Triton X in PBS for 1 h. Guinea pig anti-insulin polyclonal antibodies (final dilution 1:1,000; DakoCytomation, Glostrup, Denmark) were used as primary antibody. Cells were incubated with guinea pig anti-insulin antiserum at room temperature for 16 h and then with biotinylated anti-guinea pig IgG (Vector Laboratories, Temecula, CA). Immunoreactivity was visualized with diaminobenzidine after labeling with streptavidin-conjugated horseradish peroxidase (DakoCytomation).

Silencing of GPR40 expression using siRNAs. Three pairs of RNA oligonucleotides were designed from the rat GPR40 sequence. These regions showed no significant homology to any other known gene (analyzed using BLAST). These siRNAs, consisting of 19 nucleotides with overhangs of two T's, were obtained from Ambion. In our experiments to suppress GPR40 expression in isolated islet cells, we used the most effective siRNAs among them (target sequence of 5'-GCTTGGTCTACACTCTCCA-3', corresponding to position 126–144 of rat GPR40 mRNA). As a control scramble siRNA, we used Silencer Negative control siRNA (Ambion). Transfection of siRNA was accomplished with Lipofectamin 2000. After 6 h, transfection solution was replaced with MEM containing 10% FBS, followed by culture for 48 h.

Quantity of GPR40 expression by real-time RT-PCR. mRNAs for GPR40 and sulfonylurea receptor 1 (SUR1) were measured by extracting RNA using TRIzol reagent and generating cDNA copies using Superscript II. Quantitative real-time PCR (ABI PRISM 7900HT; Applied Biosystems, Foster City, CA) was done as follows: 40 cycles of PCR (95°C for 15 s, 60°C for 1 min) after initial denaturation for 10 min at 95°C by using 10 µl of 2x qPCR Mastermix Plus for SYBR Green I, 150 nmol primers, 0.5 µl cDNA, and H₂O to 20 µl. GPR40 and SUR1 mRNA levels were normalized by using -actin as a housekeeping gene. Primers used for real-time PCR are as follows: rat GPR40 5'-CCTTTGCACTAGGCTTTCCA-3', 5'-AGGTCAGAGCAGGCCAAAT-3'; rat SUR1, 5'-CACATTCACCACAGCACCTG-3', 5'-CCAGCTGGCATGTATAAGTG-3'; rat -actin, 5'-tggcaccacactttctacaatgagc-3', 5'-GGGTCATCTTTTCACGGTTGG-3'.

Measurement of insulin release. Measurement of insulin release was carried out as previously described (34). Briefly, groups of 10 islets were incubated at 37°C for 0.5 h in KRBH with 0.01% BSA and 2.8 mM glucose and then for 0.5 h in KRBH with 0.01% BSA and 8.3 mM glucose. OA, U-73343, U-73122, and nitrendipine were added in KRBH throughout incubation. Insulin concentration was determined using enzyme immunosorbent assay kits (Morinaga, Yokohama, Japan).

Statistical analysis. The data are presented as means ± SE. Each study was based on -cells prepared from at least three rats. The data were examined by one-way analysis of variance with repeated measurements, and differences between treatment groups were evaluated using Dunnett's multiple test. P values of <0.05 were considered statistically significant.

	RESULTS

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Glucose- and concentration-dependent effects of OA to increase [Ca²⁺]_i. Single islet cells were superfused with KRBH and subjected to [Ca²⁺]_i measurements using fura 2 fluorescence imaging. OA at 10 µM failed to increase [Ca²⁺]_i in the presence of 2.8 mM glucose, but in the presence of 11.2 mM glucose it induced rapid and large increases in [Ca²⁺]_i in a single islet cell, which was subsequently shown to be immunoreactive to insulin (Fig. 1, A and B). The result demonstrated that OA increases [Ca²⁺]_i in rat islet -cells. Repeated administration of OA induced repetitive [Ca²⁺]_i increases. Stimulation with 11.2 mM glucose and 300 µM tolbutamide also increased [Ca²⁺]_i (Fig. 1A). OA (10 µM) at 2.8 mM glucose increased [Ca²⁺]_i in none of 61 -cells (0%), at 5.6 mM glucose in 18 of 53 cells (34%), at 8.3 mM glucose in 75 of 104 cells (72%), and at 11.2 mM glucose in 239 of 274 cells (87%), showing a glucose-dependent effect (Fig. 1C). Thus OA was ineffective at a basal glucose concentration but effective at glucose concentrations that stimulate insulin release, suggesting that OA acts as a potentiator of the glucose action on [Ca²⁺]_i in rat -cells. At 11.2 mM glucose, OA (10 µM) increased [Ca²⁺]_i with latency of 44 ± 3 s after addition to the superfusion medium and with amplitude of 103 ± 3 nM (n = 239).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Glucose- and concentration-dependent effects of oleic acid (OA) to increase [Ca2+]i in single -cells. A and B: OA at 10 µM was without effect on [Ca2+]i in the presence of 2.8 mM glucose (2.8 G) but evoked increases in [Ca2+]i after the glucose concentration was elevated to 11.2 mM, in a single islet cell (A), which was proved to be immunoreactive to insulin by subsequent immunocytochemical staining with anti-insulin antiserum (B; arrowhead). Scale bar, 10 µm. The cell also responded to 11.2 mM glucose and 300 µM tolbutamide (Tolb; A). Bars above the tracing specify periods of exposure to compounds. C: percentage of -cells responding to OA at each concentration in the presence of 11.2 mM glucose. Nos. above each point indicate no. of -cells that responded over no. of -cells examined. D: OA at 3, 5, and 10 µM increased [Ca2+]i, whereas OA at 1 µM had little effect on [Ca2+]i in the presence 11.2 mM glucose. E: percentage of -cells responding to 1, 3, 5, and 10 µM OA in the presence of 11.2 mM glucose. Nos. above each point indicate no. of cells that responded over no. of cells examined. F: increases in [Ca2+]i during stimulation with 1, 3, 5, and 10 µM OA for 3 min were integrated and averaged for the cells examined. *P < 0.01 vs. 1 µM OA. G: elevation of BSA concentration to 0.1% inhibited the [Ca2+]i response to 10 µM OA. BSA concentration in superfusion solutions was 0.01% in all other experiments.

Inhibition of OA-induced [Ca²⁺]_i increases by BSA. It was reported that FFAs tightly bind to BSA (see reviews in Refs. 17 and 24). To avoid significant binding, all experiments in the present study were carried out in solutions containing BSA at a concentration as low as 0.01% unless otherwise indicated. The [Ca²⁺]_i increase induced by 10 µM OA was abolished in the presence of an elevated BSA concentration of 0.1% (Fig. 1G).

Effect of GPR40 silencing on OA-induced [Ca²⁺]_i increases. To investigate the involvement of GPR40 in the OA-induced [Ca²⁺]_i increase in -cells, endogenous GPR40 mRNA was reduced by RNA silencing. Expressions of mRNAs for GPR40 and SUR1 were measured by quantitative real-time PCR using the ABI PRISM 7900HT sequence detection system. Expressions of GPR40 and SUR1 were normalized by the mRNA expression for -actin, a housekeeping gene. These receptor mRNA levels in the cells transfected with GPR40 siRNA were expressed by the percentage of those in the negative control (NC) siRNA-transfected cells. Transfection of an siRNA duplex corresponding to nucleotides 126–144 of rat GPR40 cDNA (rGPR40 siRNA) significantly reduced GPR40 mRNA expression in isolated islet cells to the level of 13.3 ± 1.4 (n = 6, P < 0.01) after 48 h (Fig. 2A, left). In contrast, rGPR40 siRNA transfection to islets cells did not affect SUR1 mRNA expression (115.9 ± 29.5; Fig. 2A, right).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effect of rat G protein receptor GPR40 silencing on OA-induced [Ca2+]i increases. Quantitative real-time RT-PCR expression analysis of GPR40 (A) and sulfonylurea receptor-1 (SUR1; B) mRNA levels. Data are shown as %change (mean ± SE) based on negative control scrambled (NC) small interfering (si)RNA-transfected group. Expression of GPR40 mRNA was inhibited in rat GPR40 (rGPR40) siRNA-transfected cells (A), whereas expression of SUR1 mRNA was unchanged in rGPR40 siRNA-transfected cells (B). Data are expressed as means ± SE; n = 4 independent experiments for NC siRNA; n = 6 for rGPR40 siRNA, respectively. *P < 0.01. C: [Ca2+]i response to 10 µM OA in the presence of 11.2 mM glucose was observed in a NC siRNA-transfected single -cell. D: in a single -cell transfected with rGPR40-specific siRNA, [Ca2+]i response to 10 µM OA in the 11.2 mM glucose was impaired, whereas [Ca2+]i responses to 11.2 mM glucose and 300 µM tolbutamide were little affected. E: percentage of -cells responding to 10 µM OA in 11.2 mM glucose in NC siRNA- and rGPR40 siRNA-transfected -cells. Nos. above each bar indicate no. of cells responding over no. examined. F: increases in [Ca2+]i during stimulation with OA (3 min) and tolbutamide (1 min) were integrated and averaged for 43 NC siRNA-transfected and 50 rGPR40 siRNA-transfected -cells. *P < 0.01.

Effect of PLC inhibitor, Ca²⁺-free condition, and L-type Ca²⁺ channel blocker on [Ca²⁺]_i responses to OA. The OA (10 µM)-induced [Ca²⁺]_i increase was inhibited by U-73122 (1 µM), a PLC inhibitor, but was little affected by U-73343 (1 µM), a related compound without an inhibitory action on PLC, in the same -cell (Fig. 3A). In the presence of 1 µM U-73122, only 2 of 45 -cells (4%) exhibited [Ca²⁺]_i responses to 10 µM OA (Fig. 3C). In contrast, in the presence of U-73343 (1 µM), 61 of 68 -cells (90%) exhibited [Ca²⁺]_i responses to OA, exhibiting the response incidence similar to that in the control experiments. Neomycin is another inhibitor of the PLC pathway: it binds to phosphatidylinositol 4,5-bisphosphate (PIP₂) and thereby inhibits the activity of PLC. Neomycin (1.5 mM) also inhibited the OA (10 µM)-induced [Ca²⁺]_i increase (Fig. 3, B and C). Thus [Ca²⁺]_i responses to OA were inhibited by two PLC inhibitors that work through distinct mechanisms.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Effects of phospholipase C (PLC) inhibitor and L-type Ca2+ channel blocker on [Ca2+]i responses to OA. A: in the presence of 1 µM U-73122, a PLC inhibitor, the [Ca2+]i response to 10 µM OA in the presence of 11.2 mM glucose was inhibited, whereas it was little affected by the presence of 1 µM U-73343, an analog of U-73122 without inhibitory action on PLC. B: in the presence of 1.5 mM neomycin (neom), an inhibitor of PLC pathway, the [Ca2+]i response to 10 µM OA in the presence of 11.2 mM glucose was inhibited. C: percentage of -cells responding to 10 µM OA in the presence of U-73343, U-73122, or neomycin. D: OA-induced [Ca2+]i increase was inhibited by 5 µM nitrendipine (NTD), a blocker of L-type Ca2+ channels. E: [Ca2+]i response to 10 µM OA in the presence of 11.2 mM glucose was little affected by the presence of 0.2 µM thapsigargin (Tg), a blocker of endoplasmic reticulum Ca2+ pump. F: percentage of -cells responding to 10 µM OA under control and Ca2+-free conditions and in the presence of NTD or Tg. Nos. above each bar indicate no. of -cells responding over no. of -cells examined.

Effect of OA to increase insulin release from islets and its attenuation by PLC and L-type Ca²⁺ channel inhibitors. Insulin release from isolated rat islets under static incubation conditions was stimulated by 8.3 mM glucose. In the presence of 8.3 mM glucose, insulin release from islets tended to be increased by OA in a concentration range of 10–50 µM, in which the effect of 50 µM OA was significant (Fig. 4A).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effect of OA on insulin release from rat islets. A: OA at 50 µM significantly increased insulin release from isolated islets in the presence of 8.3 mM glucose. *P < 0.05 vs. 8.3 mM glucose alone. B: increased insulin release by 50 µM OA at 8.3 mM glucose was attenuated by the presence of 2 µM U-73122, but not 2 µM U-73343, and by 10 µM NTD. Results are presented as means ± SE for 6–12 experiments in each group. *P < 0.05, **P < 0.01 vs. 50 µM OA in the presence of 8.3 mM glucose without inhibitor (non).

	DISCUSSION

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The ability of FFAs to stimulate insulin secretion is well established, but the cellular mechanisms are inadequately understood. In the present study, we found that administration of OA evoked rapid increases in [Ca²⁺]_i in a concentration- and glucose-dependent manner in rat pancreatic -cells. We demonstrated, for the first time, that OA increases [Ca²⁺]_i mainly via interaction with the G protein-coupled receptor GPR40 and signaling mechanisms involving PLC and enhanced Ca²⁺ entry through L-type Ca²⁺ channels in normal rat pancreatic -cells.

In the present study, transfection with the rat GPR40 specific siRNA largely reduced GPR40 mRNA levels in isolated islet cells. In these -cells with reduced GPR40 mRNA, OA-induced [Ca²⁺]_i increases were markedly inhibited, whereas [Ca²⁺]_i responses to high glucose and tolbutamide were unchanged. This result is in accord with a previous report that the increase of insulin secretion from MIN6 cells after stimulation with FFAs was eliminated by treatment with the siRNA specific for mouse GPR40 (15). Therefore, our data demonstrate that OA increases [Ca²⁺]_i largely via interaction with GPR40 in rat -cells.

In the current study, the concentration of BSA in the superfusion medium was as low as 0.01%. Elevation of the BSA concentration to 0.1% abolished the ability of OA to increase [Ca²⁺]_i. It was previously reported that BSA at 0.1% or higher inhibited the Ca²⁺ influx induced by FFAs (10 µM) in human GPR40-expressing CHO cells (15). Therefore, it is likely that the free, but not the bound, form of OA is capable of interacting with GPR40 to increase [Ca²⁺]_i in rat pancreatic -cells.

The present study demonstrated that OA-induced [Ca²⁺]_i increases were inhibited by the PLC inhibitor U-73122 but not its analog without PLC-inhibiting activity, and by neomycin, which counteracts the PLC pathway in rat pancreatic -cells. The result suggests that the OA-stimulated, GPR40-mediated increase in [Ca²⁺]_i depends on PLC in rat pancreatic -cells. GPR40 is a member of the G protein-coupled receptor superfamily (22). The -subunit of the G_q family of G proteins (G_q) is well known to stimulate PLC activity (23, 28). Therefore, GPR40 of rat pancreatic -cells may be linked to PLC activation via G proteins, particularly G_q, in response to OA stimulation. This concept is consistent with previous reports in cell lines: human GPR40 (hGPR40) is coupled to G_q (3) or to both G_q and G_i (15) in the hGPR40-expressing CHO cells, and hGPR40 is coupled to G_i/o and G_q in the human breast cancer cell line MCF-7 (36). Taken together, it is suggested that rat GPR40 is probably coupled with G_q and thereby with PLC signaling cascades.

Furthermore, both the extracellular Ca²⁺-free condition and addition of an L-type Ca²⁺ channel blocker inhibited OA-induced [Ca²⁺]_i increases, whereas thapsigargin, a blocker of the endoplasmic reticulum Ca²⁺ pump, had no effect on them. These data suggest that OA stimulates Ca²⁺ influx through L-type Ca²⁺ channels in -cells. However, the signaling mechanism that causes the opening of L-type Ca²⁺ channels is yet unknown. It is known that acetylcholine (ACh) stimulates insulin secretion via G_q-coupled musucarinic 3 receptor. ACh depolarizes the -cell plasma membrane by increasing its permeability to Na⁺. This depolarization facilitates the voltage-dependent L-type Ca²⁺ channel that is initially activated by glucose and other secretagogues that depolarize the -cell plasma membrane (9, 10, 13, 30). Although most of the studies on ACh-induced Na⁺ entry have been done using mouse -cells, functional Na⁺ channels are also located in rat -cells and involved in insulin secretion (14). Therefore, the Na⁺-dependent mechanism reported for the ACh signaling pathway could also operate in the [Ca²⁺]_i responses to FFAs in rat -cells. However, further studies are needed to clarify the signal transduction mechanisms that link the activation of GPR40 to the voltage-dependent Ca²⁺ influx and possibly insulin secretion in OA-stimulated -cells.

It is well established that a [Ca²⁺]_i increase triggers insulin secretion from -cells. We found that OA increased both [Ca²⁺]_i in -cells and insulin secretion in isolated islets. Moreover, the OA-induced [Ca²⁺]_i increase and insulin release were both markedly attenuated by inhibitors of PLC and L-type Ca²⁺ channels. These results strongly suggest that the PLC- and L-type Ca²⁺ channel-mediated [Ca²⁺]_i increase is linked to insulin release in OA-stimulated islet -cells. In the present study, the concentration of OA required for insulin release was 5–10 times higher than that for [Ca²⁺]_i increase, and extents of inhibition by PLC and Ca²⁺ channel blockers were smaller for insulin release than for [Ca²⁺]_i increase. This may be due to differences in experimental conditions employed: [Ca²⁺]_i was measured in single -cells whereas insulin release was measured in islets. In the former case, -cells are expected to see OA and inhibitors at the concentrations added in superfusion solution. By contrast, in islets in which non--cells are located in the periphery and -cells in the core, OA and inhibitors have to enter the core of islets, and therefore the actual concentrations at which they act on -cells may be lower than those originally added in solution. If so, it is expected that, when the same concentrations are used, the effects of OA and inhibitors are weaker in islets than in single -cells and that higher concentrations of OA are needed in the islet experiments to get comparable effects. Alternatively, unknown factors that are released in islets and modulate the effects of OA on -cells could be involved in the islet experiments.

Taken together, the present findings suggest that the OA-induced GPR40-mediated increase in [Ca²⁺]_i is linked to insulin release. In support of this concept, Steneberg et al. (27) recently reported a study on GPR40-deficient mice, in which palmitic acid-stimulated insulin secretion was blunted in islets isolated from GPR40-null mice. Additionally, the OA-induced [Ca²⁺]_i increase could be related to -cell functions other than stimulation of insulin release. A possibility is that long-term exposure to OA could induce long-lasting elevation of [Ca²⁺]_i, thereby evoking the Ca²⁺ toxicity and consequent dysfunction of islet -cells.

At a basal glucose concentration of 2.8 mM, OA even at the highest concentration (10 µM) examined had no effect on [Ca²⁺]_i, whereas at 5.6 mM glucose it increased [Ca²⁺]_i in 30% and at 11.2 mM glucose in 90% of -cells. Thus the concentration of glucose above which it permits the effect of OA appears to be in the range somewhat lower than 5.6 mM, which may be close to the threshold glucose concentration for initiating insulin release in rats. The glucose-dependent property of the effect of FFAs was also previously reported (15): FFAs stimulated insulin secretion in MIN6 cells and rat islets at glucose concentrations over 11 mM, and OA (>3 µM) significantly stimulated insulin secretion in MIN6 cells at glucose concentration of 22 mM. Thus a strictly glucose-dependent ability of FFAs to increase [Ca²⁺]_i and insulin release has been observed in rat -cells and MIN6 cells, although the threshold glucose concentration that permits FFAs to be effective is different in each preparation. This property that the effect of OA is turned on when the glucose concentration is elevated to the stimulatory range appears to be physiologically relevant: the circulating level of FFAs increases upon prolonged fasting, but it cannot influence insulin release from -cells at the fasting low plasma glucose levels, providing a safe mechanism to prevent insulin release and consequent hypoglycemia. However, when plasma glucose is increased in response to meals, it may be able to synergize with the already elevated levels of FFAs to yield a sufficient activation of -cells and insulin release, thereby promoting an effective transport of glucose and lipids into the organs that are short of energy reserves. However, further studies are definitely needed to clarify the roles of GPR40 in insulin release in both physiological and pathophysiological states. Furthermore, the relative contributions and possible interaction of the GPR40-mediated and metabolism-mediated pathways in the reception and signaling of FFAs and their link to insulin release in islet -cells remain to be clarified.

	GRANTS

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This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Priority Areas (15081101) from JSPS, and a grant from the 21st century COE program to T. Yada.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. K. Dezaki and M. Nakata for technical advice and valuable discussion, and thank Y. Nishizawa and S. Ohkuma for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Yada, Dept. of Physiology, Div. of Integrative Physiology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan (e-mail: tyada{at}jichi.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Alcazar O, Qiu-yue Z, Gine E, and Tamarit-Rodriguez J. Stimulation of islet protein kinase C translocation by palmitate requires metabolism of the fatty acid. Diabetes 46: 1153–1158, 1997.[Abstract]
2. Balasse EO and Ooms HA. Role of plasma free fatty acids in the control of insulin secretion in man. Diabetologia 9: 145–151, 1973.[CrossRef][Web of Science][Medline]
3. Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, Ellis C, Elshourbagy NA, Goetz AS, Minnick DT, Murdock PR, Sauls, HR Jr, Shabon U, Spinage LD, Strum JC, Szekeres PG, Tan KB, Way JM, Ignar DM, Wilson S, and Muir AI. The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem 278: 11303–11311, 2003.[Abstract/Free Full Text]
4. Corkey BE, Deeney JT, Yaney GC, Tornheim K, and Prentki M. The role of long-chain fatty acyl-CoA esters in beta-cell signal transduction. J Nutr 130: 299S–304S, 2000.[Abstract/Free Full Text]
5. Crespin SR, Greenough WB III, and Steinberg D. Stimulation of insulin secretion by infusion of free fatty acids. J Clin Invest 48: 1934–1943, 1969.[Web of Science][Medline]
6. Deeney JT, Gromada J, Hoy M, Olsen HL, Rhodes CJ, Prentki M, Berggren PO, and Corkey BE. Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells). J Biol Chem 275: 9363–9368, 2000.[Abstract/Free Full Text]
7. Deeney JT, Tornheim K, Korchak HM, Prentki M, and Corkey BE. Acyl-CoA esters modulate intracellular Ca²⁺ handling by permeabilized clonal pancreatic beta-cells. J Biol Chem 267: 19840–19845, 1992.[Abstract/Free Full Text]
8. Dobbins RL, Chester MW, Daniels MB, McGarry JD, and Stein DT. Circulating fatty acids are essential for efficient glucose-stimulated insulin secretion after prolonged fasting in humans. Diabetes 47: 1613–1618, 1998.[Abstract]
9. Gagerman E, Sehlin J, and Taljedal IB. Effects of acetylcholine on ion fluxes and chlorotetracycline fluorescence in pancreatic islets. J Physiol 300: 505–513, 1980.[Abstract/Free Full Text]
10. Gilon P, Nenquin M, and Henquin JC. Muscarinic stimulation exerts both stimulatory and inhibitory effects on the concentration of cytoplasmic Ca²⁺ in the electrically excitable pancreatic B-cell. Biochem J 311: 259–267, 1995.
11. Gravena C, Mathias PC, and Ashcroft SJ. Acute effects of fatty acids on insulin secretion from rat and human islets of Langerhans. J Endocrinol 173: 73–80, 2002.[Abstract]
12. Greenough WB III, Crespin SR, and Steinberg D. Hypoglycaemia and hyperinsulinaemia in response to raised free-fatty-acid levels. Lancet 2: 1334–1336, 1967.[Web of Science][Medline]
13. Henquin JC, Garcia MC, Bozem M, Hermans MP, and Nenquin M. Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx. Endocrinology 122: 2134–2142, 1988.[Abstract/Free Full Text]
14. Hiriart M, Matteson DR. Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay. J Gen Physiol 91: 617–639, 1988.[Abstract/Free Full Text]
15. Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H, Tanaka H, Maruyama M, Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura F, Noguchi Y, Shinohara T, Hinuma S, Fujisawa Y, and Fujino M. Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature 422: 173–176, 2003.[CrossRef][Medline]
16. Kotarsky K, Nilsson NE, Flodgren E, Owman C, and Olde B. A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs. Biochem Biophys Res Commun 301: 406–410, 2003.[CrossRef][Web of Science][Medline]
17. Kragh-Hansen U. Molecular aspects of ligand binding to serum albumin. Pharmacol Rev 33: 17–53, 1981.[Web of Science][Medline]
18. Larsson O, Deeney JT, Branstrom R, Berggren PO, and Corkey BE. Activation of the ATP-sensitive K⁺ channel by long chain acyl-CoA. A role in modulation of pancreatic beta-cell glucose sensitivity. J Biol Chem 271: 10623–10626, 1996.[Abstract/Free Full Text]
19. Mason TM, Goh T, Tchipashvili V, Sandhu H, Gupta N, Lewis GF, and Giacca A. Prolonged elevation of plasma free fatty acids desensitizes the insulin secretory response to glucose in vivo in rats. Diabetes 48: 524–530, 1999.[Abstract]
20. Moore PC, Ugas MA, Hagman DK, Parazzoli SD, and Poitout V. Evidence against the involvement of oxidative stress in fatty acid inhibition of insulin secretion. Diabetes 53: 2610–2616, 2004.[Abstract/Free Full Text]
21. Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, and Corkey BE. Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267: 5802–5810, 1992.[Abstract/Free Full Text]
22. Sawzdargo M, George SR, Nguyen T, Xu S, Kolakowski LF, and O'Dowd BF. A cluster of four novel human G protein-coupled receptor genes occurring in close proximity to CD22 gene on chromosome 19q13.1. Biochem Biophys Res Commun 239: 543–547, 1997.[CrossRef][Web of Science][Medline]
23. Smrcka AV, Hepler JR, Brown KO, and Sternweis PC. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251: 804–807, 1991.[Abstract/Free Full Text]
24. Spector AA. Fatty acid binding to plasma albumin. J Lipid Res 16: 165–179, 1975.[Abstract]
25. Stein DT, Esser V, Stevenson BE, Lane KE, Whiteside JH, Daniels MB, Chen S, and McGarry JD. Essentiality of circulating fatty acids for glucose-stimulated insulin secretion in the fasted rat. J Clin Invest 97: 2728–2735, 1996.[Web of Science][Medline]
26. Stein DT, Stevenson BE, Chester MW, Basit M, Daniels MB, Turley SD, and McGarry JD. The insulinotropic potency of fatty acids is influenced profoundly by their chain length and degree of saturation. J Clin Invest 100: 398–403, 1997.[Web of Science][Medline]
27. Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD, and Edlund H. The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metabolism 1: 245–258, 2005.
28. Taylor SJ, Chae HZ, Rhee SG, and Exton JH. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature 350: 516–518, 1991.[CrossRef][Medline]
29. Warnotte C, Gilon P, Nenquin M, and Henquin JC. Mechanisms of the stimulation of insulin release by saturated fatty acids. A study of palmitate effects in mouse beta-cells. Diabetes 43: 703–711, 1994.[Abstract]
30. Yada T, Hamakawa N, and Yaekura K. Two distinct modes of Ca²⁺ signalling by ACh in rat pancreatic beta-cells: concentration, glucose dependence and Ca²⁺ origin. J Physiol 488: 13–24, 1995.[Abstract/Free Full Text]
31. Yada T, Itoh K, and Nakata M. Glucagon-like peptide-1-(7–36)amide and a rise in cyclic adenosine 3',5'-monophosphate increase cytosolic free Ca²⁺ in rat pancreatic beta-cells by enhancing Ca²⁺ channel activity. Endocrinology 133: 1685–1692, 1993.[Abstract/Free Full Text]
32. Yada T, Kakei M, and Tanaka H. Single pancreatic beta-cells from normal rats exhibit an initial decrease and subsequent increase in cytosolic free Ca²⁺ in response to glucose. Cell Calcium 13: 69–76, 1992.[CrossRef][Web of Science][Medline]
33. Yada T, Sakurada M, Ihida K, Nakata M, Murata F, Arimura A, and Kikuchi M. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet beta-cells. J Biol Chem 269: 1290–1293, 1994.[Abstract/Free Full Text]
34. Yada T, Sakurada M, Ishihara H, Nakata M, Shioda S, Yaekura K, Hamakawa N, Yanagida K, Kikuchi M, and Oka Y. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats. J Physiol 505: 319–328, 1997.[Abstract/Free Full Text]
35. Yaney GC, Korchak HM, and Corkey BE. Long-chain acyl CoA regulation of protein kinase C and fatty acid potentiation of glucose-stimulated insulin secretion in clonal beta-cells. Endocrinology 141: 1989–1998, 2000.[Abstract/Free Full Text]
36. Yonezawa T, Katoh K, and Obara Y. Existence of GPR40 functioning in a human breast cancer cell line, MCF-7. Biochem Biophys Res Commun 314: 805–809, 2004.[CrossRef][Web of Science][Medline]
37. Zhou YP and Grill VE. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93: 870–876, 1994.[Web of Science][Medline]

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