It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic β-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 β-cells are little known. The present study was aimed at elucidating GPR40-linked Ca2+ signaling mechanisms in rat pancreatic β-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 β-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 β-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 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 β-cells, which may be link to insulin release.
- G protein-coupled receptor
- intracellular calcium
- insulin secretion
- free fatty acids
- phospholipase C
long-chain free fatty acids (FFAs), as well as glucose, are the principal energy sources for the body. FFAs are also considered the systemic signals that link metabolic states to insulin release. Both stimulatory and inhibitory effects of FFAs on insulin secretion have been documented. In the short term, acute increases in circulating FFAs promote insulin secretion from pancreatic β-cells (2, 5, 11, 12, 26). By contrast, when the plasma level of FFAs is chronically elevated, it inhibits insulin secretion (19, 20, 37), a phenomenon recognized as lipotoxicity.
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 β-cell lines (21, 29). An increase in cytosolic LC-CoA activates or modulates various processes in β-cells: activation of protein kinase C (PKC) (1, 35), modulation of ATP-sensitive potassium (KATP) 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 β-cells by in situ hybridization (15). A significant amount of GPR40 mRNA is also detected in β-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 β-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 β-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 β-cells, which may be linked to insulin release.
MATERIALS AND METHODS
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 β-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 β-cells that were selected by immunostaining with anti-insulin antiserum or by morphological and physiological criteria for these β-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.
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 2× 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 β-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 β-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).
The data are presented as means ± SE. Each study was based on β-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.
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 β-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 β-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 β-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).
In the presence of 11.2 mM glucose, OA at 1 μM increased [Ca2+]i in 25 of 73 cells (34%), at 3 μM in 21 of 29 cells (72%), at 5 μM in 59 of 77 cells (77%), and at 10 μM in 239 of 274 cells (87%), showing a concentration-dependent effect (Fig. 1, D and E). The OA-evoked increase in [Ca2+]i above the basal level (Δ[Ca2+]i) was integrated over the 3-min administration period and expressed by the amount per minute. OA increased the integrated Δ[Ca2+]i in a concentration-dependent manner (Fig. 1F).
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 β-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 β-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).
NC siRNA-transfected β-cells responded to 11.2 mM glucose, 10 μM OA, and tolbutamide (Fig. 2B). In contrast, in rGPR40 siRNA-transfected β-cells, the [Ca2+]i response to 10 μM OA was severely impaired, whereas responses to both 11.2 mM glucose and tolbutamide were normally observed (Fig. 2C). OA at 10 μM increased [Ca2+]i only in 4 of 50 rGPR40 siRNA-transfected β-cells (7%), whereas it increased [Ca2+]i in 33 of 43 NC siRNA-transfected β-cells (77%; Fig. 2D). The integrated Δ[Ca2+]i during exposure to OA in rGPR40 siRNA-transfected β-cells was significantly lower than that in NC siRNA-transfected β-cells (85 ± 15 nM/min, n = 50, for rGPR40 siRNA vs. 331 ± 37 nM/min, n = 43, for NC siRNA, P < 0.05; Fig. 2E). By contrast, the integrated Δ[Ca2+]i during exposure to tolbutamide for 1 min was not different between rGPR40 siRNA- and NC siRNA-transfected β-cells (406 ± 44 nM/min, n = 50, for rGPR40 siRNA vs. 532 ± 64 nM/min, n = 43, for NC siRNA; Fig. 2E). Thus both the incidence and the magnitude of the [Ca2+]i response to OA were selectively and severely inhibited by transfection of rGPR40 siRNA, suggesting that major parts, if not all, of the [Ca2+]i response to OA are mediated by GPR40 in rat pancreatic β-cells.
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 β-cell (Fig. 3A). In the presence of 1 μM U-73122, only 2 of 45 β-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 β-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.
The OA (10 μM)-induced [Ca2+]i increase was inhibited by an L-type Ca2+ channel blocker, nitrendipine (5 μM; Fig. 3D). The [Ca2+]i increase in response to OA (10 μM) was observed in none of 30 β-cells in the presence of 5 μM nitrendipine and in none of 26 β-cells under Ca2+-free conditions made with 0.1 mM EGTA and no added Ca2+ (Fig. 3F). In contrast, a blocker of the endoplasmic reticulum Ca2+ pump, thapsigargin (0.2 μM), had no effect on the OA-induced [Ca2+]i increase (Fig. 3, E and F).
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).
The increase in insulin release in response to OA (50 μM) at 8.3 mM glucose was substantially attenuated by U-73122 (2 μM), a PLC inhibitor, whereas U-73343 (2 μM), a related compound without an inhibitory action on PLC, had no effect (Fig. 4B). Furthermore, nitrendipine (10 μM), an L-type Ca2+ channel blocker, also reduced the OA-induced increase in insulin release (Fig. 4B).
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 β-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 β-cells.
In the present study, transfection with the rat GPR40 specific siRNA largely reduced GPR40 mRNA levels in isolated islet cells. In these β-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 β-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 β-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 β-cells. The result suggests that the OA-stimulated, GPR40-mediated increase in [Ca2+]i depends on PLC in rat pancreatic β-cells. GPR40 is a member of the G protein-coupled receptor superfamily (22). The α-subunit of the Gq family of G proteins (Gqα) is well known to stimulate PLC activity (23, 28). Therefore, GPR40 of rat pancreatic β-cells may be linked to PLC activation via G proteins, particularly Gqα, in response to OA stimulation. This concept is consistent with previous reports in cell lines: human GPR40 (hGPR40) is coupled to Gqα (3) or to both Gqα and Giα (15) in the hGPR40-expressing CHO cells, and hGPR40 is coupled to Gi/oα and Gqα in the human breast cancer cell line MCF-7 (36). Taken together, it is suggested that rat GPR40 is probably coupled with Gqα 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 β-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 Gqα-coupled musucarinic 3 receptor. ACh depolarizes the β-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 β-cell plasma membrane (9, 10, 13, 30). Although most of the studies on ACh-induced Na+ entry have been done using mouse β-cells, functional Na+ channels are also located in rat β-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 β-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 β-cells.
It is well established that a [Ca2+]i increase triggers insulin secretion from β-cells. We found that OA increased both [Ca2+]i in β-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 β-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 β-cells whereas insulin release was measured in islets. In the former case, β-cells are expected to see OA and inhibitors at the concentrations added in superfusion solution. By contrast, in islets in which non-β-cells are located in the periphery and β-cells in the core, OA and inhibitors have to enter the core of islets, and therefore the actual concentrations at which they act on β-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 β-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 β-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 β-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 β-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 β-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 β-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 β-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 β-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 β-cells remain to be clarified.
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.
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.
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- Copyright © 2005 by American Physiological Society