Reduction in Voltage-Gated K+ Currents in Primary Cultured Rat Pancreatic -Cells by Linoleic Acids
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《内分泌学杂志》
Prince Henry’s Institute of Medical Research (D.D.F., M.H., N.T., D.J.K., C.C.), Clayton, Victoria 3168, Australia
Department of Physiology (D.D.F., Z.L.), XiangYa Medical School, Central-South University, Hunan 410018, China
Department of Food Production Science (S.R.), Shinshu University, Nagano-ken 399-4598, Japan
Abstract
Free fatty acids (FFAs), in addition to glucose, have been shown to stimulate insulin release through the G protein-coupled receptor (GPCR)40 receptor in pancreatic -cells. Intracellular free calcium concentration ([Ca2+]i) in -cells is elevated by FFAs, although the mechanism underlying the [Ca2+]i increase is still unknown. In this study, we investigated the action of linoleic acid on voltage-gated K+ currents. Nystatin-perforated recordings were performed on identified rat -cells. In the presence of nifedipine, tetrodotoxin, and tolbutamide, voltage-gated K+ currents were observed. The transient current represents less than 5%, whereas the delayed rectifier current comprises more than 95%, of the total K+ currents. A long-chain unsaturated FFA, linoleic acid (10 μM), reversibly decreased the amplitude of K+ currents (to less than 10%). This reduction was abolished by the cAMP/protein kinase A system inhibitors H89 (1 μM) and Rp-cAMP (10 μM) but was not affected by protein kinase C inhibitor. In addition, forskolin and 8'-bromo-cAMP induced a similar reduction in the K+ current as that evoked by linoleic acid. Insulin secretion and cAMP accumulation in -cells were also increased by linoleic acid. Methyl linoleate, which has a similar structure to linoleic acid but no binding affinity to GPR40, did not change K+ currents. Treatment of cultured cells with GPR40-specific small interfering RNA significantly reduced the decrease in K+ current induced by linoleic acid, whereas the cAMP-induced reduction of K+ current was not affected. We conclude that linoleic acid reduces the voltage-gated K+ current in rat -cells through GPR40 and the cAMP-protein kinase A system, leading to an increase in [Ca2+]i and insulin secretion.
Introduction
IT IS APPARENT that excessive fat tissue or obese conditions facilitate the progress of type 2 diabetes and may be responsible for the dramatic increase in the occurrence of diabetes in recent years. Abnormal metabolism of carbohydrate and lipid has been found in type 2 diabetes with reduced efficacy of insulin (insulin resistance) in muscle, liver, and other tissues (1, 2). Also, the release of insulin from pancreatic -cells in response to increased blood glucose is elevated in the early stage of diabetes to compensate insulin resistance. Free fatty acids (FFAs) are mainly released from adipose tissue as an energy source, and the acute elevation of plasma FFAs is necessary for insulin secretion (3). The detailed mechanism for such stimulation is, however, not clearly understood.
Recent reports provide evidence that long-chain FFAs are specific ligands of an orphan G protein-coupled receptor (GPCR), GPR40 (4, 5). GPR40 is abundantly expressed in pancreatic -cells and insulin-secreting cell lines and its structure is highly conserved. Functionally, FFAs increase insulin release and amplify glucose-stimulated insulin secretion through an increase in intracellular free calcium concentration ([Ca2+]i), via both Ca2+ influx through Ca2+ channels and Ca2+ release from intracellular storage sites in cultured -cell lines (4).
Regulation of insulin secretion by glucose involves the coordinated control of ion channels in the -cell membrane (6). The initial response of -cells to glucose is closure of the ATP-sensitive K+ channels. This leads to membrane depolarization followed by the activation of Ca2+ influx through voltage-gated Ca2+ channels (6). In addition, voltage-gated outward K+ currents have been detected in -cells and are demonstrated to mediate membrane potential repolarization and limit Ca2+ influx and insulin secretion (7, 8, 9, 10). It is not yet established whether voltage-gated K+ channels are directly affected by glucose and FFAs.
In the present study, we characterized voltage-gated K+ currents and demonstrated the effect of the unsaturated long-chain FFA, linoleic acid, on K+ currents in primary cultured rat pancreatic -cells. We further elucidated possible receptor and signaling systems employed by linoleic acid in the MIN6 -cell line using small interfering (si) RNA and biochemical assays. Our data suggest that such an effect is mediated by GPR40 and the cAMP-protein kinase A (PKA) signal transduction system.
Materials and Methods
Chemical reagents
Linoleic acid and methyl linoleate were purchased from Sigma (St. Louis, MO). Histopaque-1077, protease (Dispase), and collagenase type V were also obtained from Sigma. RPMI 1640, DMEM, HEPES, and carbohydrate solution was purchased from Thermo Electron (Melbourne, Victoria, Australia). Nifedipine, tolbutamide, nystatin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, 8'-bromo-cAMP and all general salts for recording solutions and molecular biological studies were purchased from Sigma. Tetrodotoxin was purchased from Alomone Laboratories (Jerusalem, Israel). Chelerythrine chloride, adenosine-3',5'-cyclic phosphorothiolate-RP (Rp-cAMP), and N-(2-(p-vromo-cinnamyl-amino)-ethyl)-5-iso-quinoline-sulfonamide (H89) were purchased from CalBiochem (Alexandria, New South Wales, Australia).
Cell culture and preparation
Pancreatic islets were isolated mostly from male Sprague Dawley rats, with an additional few from male Wistar rats (8–10 wk). We compared -cells from two strains and did not find any differences in the K+ current and the K+ current response to linoleic acid. After the pancreas was injected with 10 ml collagenase solution (1.5 mg/ml collagenase in Hank’s solution) through the common bile duct, the tissue was incubated at 37 C for 30 min. The digested pancreas was then washed, filtered through 355 μm mesh, and resuspended in cold RPMI 1640 medium. Islets were isolated using a density gradient created by different concentrations of histopaque-1077 in RPMI 1640 medium after centrifugation (11). Purified islets were further dispersed into single cells by digestion with 1 mg/ml Dispase for 10 min at room temperature. Cells were then seeded into 35-mm dishes in RPMI 1640 medium supplemented by 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured in a humidified atmosphere containing 5% CO2 at 37 C. The culture medium was replenished every 2 d, and all electrophysiological recordings were performed on the rat pancreatic -cells cultured for 2–10 d. Pancreatic -cells accounted for over 70% of total cultured cells and were identified by their significantly larger size. In addition, membrane depolarization by 15 mM glucose was tested in more than 20 cells in early establishment of recording system on primary cultured islet cells to confirm that the recorded cells were -cells. The K+ current and response to linoleic acid in the cells cultured over different lengths of time were carefully evaluated, and no difference was found. The MIN6 cell line (Walter and Eliza Institute, Melbourne, Australia) is a mouse pancreatic -cell line that retains the morphological characteristics of the pancreatic -cells (12). These cells were routinely cultured in 25 mM glucose DMEM + 10% FBS + 1% penicillin/streptomycin medium.
RNA isolation and RT-PCR
The total RNA from cells (1 x 106cells/dish) and rat islets (300 islets/dish) cultured in 35-mm petri dishes was extracted by established Trizol method (13). The total RNA from mouse heart and kidney tissue is from another laboratory in the institute. One microgram of total RNA was then reverse transcribed to cDNA in a 20-μl reverse transcription (RT) reaction system containing random primers and avian myeloblastosis virus reverse transcriptase. The RT reactions were carried out at 46 C for 2 h. One microgram of the RT reaction product was used for subsequent PCR amplification. Primer for rat GPR40 was designed according to the rat cDNA templates with an expected size of 79 bp. The set of primers for the amplification of the GPR40 was 5'-CCTATAATGCTTCCAATGTGGCTAGT-3' (forward) and 5'-CCTGTGATGAGCCCCAACTT-3' (reverse) (14). We used a denaturation step at 95 C for 30 sec, an annealing step at 60 C for 30 sec, and an extension step at 72 C for 30 sec for a total of 25 cycles, followed by an additional extension step at 72 C for 5 min. Detection of PCR amplification products was carried out by electrophoresis (1.4% agarose gel containing ethidium bromide).
Electrophysiological recording
On the day of recording, culture medium was discarded and cells were incubated in bath solution for 10–20 min before recording. Transmembrane currents were recorded using the nystatin-perforated method in whole cell recording configuration (15, 16). Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Equipment Co., Navato, CA) from borosilicate micropipettes with inner filament. After fire polishing, electrodes had an initial tip resistance of 5–8 M. All recordings were made using the Axopatch 200 amplifier and pClamp7 software (Axon Instruments, Foster City, CA).
The standard bath solution was composed of the following (millimoles): 140 NaCl, 5 KCl, 2.5 CaCl2, 0.5 MgCl2, 5 glucose, and 10 HEPES (pH 7.4) and osmolarity of 310 mOsm. The pipette solution contained (millimoles): 55 KCl, 75 K2SO4, 8 MgSO4, and 10 HEPES (pH7.4) and osmolarity of 300 mOsm. The electrode was backfilled with this solution containing nystatin (200 μg/ml in 0.3% dimethyl sulfoxide). After obtaining a high-resistance seal, the holding potential was set to –80 mV and voltage pulses (30 mV, 200 msec) were delivered periodically to monitor the appearance of a membrane capacitance transient current, which usually occurred within 3–5 min under the experimental conditions, indicating formation of a low-resistance access to the cell. Typically the series resistance reached a steady level less than 35 M within 5 min of making a seal. Input resistance of cells was higher than 10 G under our recording conditions. Whole-cell capacitance (30 cells, 8 ± 0.42 pF) and series resistance were compensated, and leak current was routinely subtracted using the option offered by Clampex program. Isolation of voltage-gated K+ currents involved the exclusion of Ca2+ (1 μM nifedipine) and Na+ (1 μM tetrodotoxin) and ATP-sensitive K+ (0.1 mM tolbutamide) currents by adding the specific blockers of these channels into the bath solution before the recording.
Original cell culture dishes were placed on the stage of an inverted microscope (Olympus, Tokyo, Japan) and cells were continuously perfused using gravity-flow at a rate of 1 ml/min. Tested FFAs and intracellular signaling stimulators were applied to the same gravity system by switching solution reservoirs. Specific intracellular signaling inhibitors were applied by hand carefully with pipette and leave for more than 10 min without perfusion to reach calculated final concentration as indicated in Results section before starting perfusion of FFAs in presence of same concentration of inhibitors onto the recorded cell. Control studies were performed by applying vehicle instead of the drugs. Because -cells were reported to be 3-fold larger than -cells (17), only cells with a diameter larger than 10 μm and well-preserved granulations were used for the recording. Recordings were low-pass filtered at 5 kHz and stored on the hard disk of an IBM-compatible computer (IBM, White Plains, NY). The value of currents was measured separately around 30 msec and 200 msec of each evoked current trace for peak (total K+ current) or steady-state [delayed rectifying K+ current (IK) current only] current during 200-msec depolarizing pulses. All experiments were performed at room temperature (20–22 C).
Intracellular cAMP and insulin secretion
MIN6 insulinoma cells (passage 28–35) were cultured in 25 mM glucose DMEM with 10% FBS, and 1% penicillin/streptomycin at 37 C and 5% CO2. Two days before cAMP sample collection and insulin secretion experiments, cells were trypsinized and plated in 24-well plates at 5 x 105 cells/well. On the day of experiment, cells were washed and preincubated in Krebs-Ringer bicarbonate (KRB) buffer for 1 h.
For cAMP measurement, cells were extracted in 1 ml of acid ethanol on top of dry ice, and extracts were dried down in a vacuum centrifuge (Speed Vac, Savant, Hicksville, NY) and stored at –20 C until assayed. cAMP in the extracts was reconstituted and measured by a sensitive and specific RIA (18).
Insulin secretion experiments were performed in KRB buffer. The cells were treated for 90 min with FFAs dissolved in KRB. The amount of insulin in the culture supernatant was determined with an ELISA kit (Diagnostic Systems Laboratories, Webster, TX).
siRNA treatment procedure in cultured MIN6 cells
RNA oligonucleotides were designed targeting AA(N19) sequences in the open reading frame of mRNA encoding mouse GPR40 as described by Itoh et al. (4). Selected siRNA target sequences were also submitted to BLAST searches against other mouse genome sequences to ensure target specificity. The following siRNA (target sequences 5'-CGCCAGUUGUGACAUUCUUdTdT-3' and 5'-AAGAAUGUCACAACUGGCGdTdT-3') was the most potent in silencing mouse GPR40-GFP proteins expressed in CHO cells (4) and was therefore used in this experiment. Scrambled siRNA duplex was used as a control (4). MIN6 cells were plated in 35-mm dishes for 2 d before transfection. Transfection of siRNA was accomplished with HVJ envelope vector kit GenomONE (Ishihara Sangyo Kaisha, Tokyo, Japan) according to the manufacturer’s instructions. Cells were incubated at 37 C in a CO2 incubator, and the culture medium was replaced 24 h after transfection. Two days after transfection, MIN6 cells were collected for RNA isolation and the GPR40 RT-PCR experiment as well as electrophysiological recording.
Statistical analysis
pClamp software (version 8.2; Axon Instruments) was used to acquire data during the recording and analyze the patch-clamp data afterward. Student’s paired t test was used as appropriate to evaluate the statistical significance of differences between treatment and control group means, and the effect was considered to be significant at the level of less than 5%. Group data represent at least four recordings under the same experimental conditions and are expressed as mean ± SEM in Results.
Results
Identification of GPR40 mRNA expression in pancreatic islets and -cells
GPR40 mRNA expression was determined in pancreatic islets and -cells by RT-PCR. Rat pancreatic islets, Min6, and INS-1 cell lines have high levels of expression of GPR40 mRNA, whereas the expression of GPR40 mRNA was detected in mouse heart tissue but undetectable under the same PCR conditions in mouse kidney tissue and GH3 rat pituitary cell line, which were used as negative controls (Fig. 1). The size of amplified fragments of the receptor was 79 bp.
Characterization of the voltage-gated K+ currents
Voltage-gated K+ currents are mainly composed of delayed IK and transient outward (IA) currents. According to the biophysical characters, currents are evoked when the cell is depolarized from different holding potentials of –40 or –80 mV, respectively. Kinetically after activation, IA is rapidly inactivated, whereas IK is noninactivated or slow inactivated. To characterize the two subtypes of voltage-gated K+ currents, depolarizing steps from two holding potentials of –80 mV (Fig. 2A) and –40 mV (Fig. 2B) were applied, in the presence of the Ca2+, Na+ and ATP-sensitive K+ channel blockers (see Materials and Methods). According to voltage-dependent inactivation properties (19), the current generated from a holding potential of –80 mV contains both IA and IK as total currents (Fig. 2A), whereas the current evoked from a holding potential of –40 mV contains only the IK current (Fig. 2B). Subtraction of currents recorded with the holding potential of –40 mV from total currents with the holding potential of –80 mV using pClamp software shows a very small portion of currents left (Fig. 2C), which represents the IA current. It is obvious that IA is very small and almost undetectable in this recording (less than 5% of total K+ current in all cells recorded). The IA current therefore represents only a minor proportion of the total voltage-gated K+ currents in this recording condition in primary cultured rat -cells, whereas the major part is IK current.
Effect of FFAs on the voltage-gated K+ current
Depolarizing test pulses from a holding potential of –80 mV elicited the outward K+ current (Fig. 3Aa), mainly IK current. Local application of 10 μM linoleic acid [the dose chosen according to published data on insulin secretion (4)] in bath solution induced a significant decrease in the amplitude of the IK current. Levels of FFAs in plasma are 0.2–1.7 mM, with 99% of these FFAs bound tightly with serum albumin (4). The concentration of unbound FFAs is in the range of 0.01–10 μM. In addition, it has been reported that unbound FFAs act on GPR40 to release insulin and the effective dose was 10 μM (4). In this context, the concentration of linoleic acid used (10 μM) in this report is physiologically relevant. Because the current now inactivated rapidly, it seems that either linoleic acid accelerated the inactivation speed of the current or the induced reduction in the current depended on the open/close gating statues of the IK channel as reported previously (20) (Fig. 3Ab). After removal of linoleic acid for 10–20 min, the K+ current recovered to the control level (Fig. 3Ac). The current was measured at the peak value (30 msec) and end (200 msec) of pulse to obtain the current-voltage relationship for both time points (Fig. 3B). From a group of eight cells, such a reduction in K+ current by linoleic acid was statistically significant at both time points (P < 0.01, Fig. 3C). We also examined oleic acid and found a similar effect on the K+ current, although the reduction was not as remarkable as that reduced by linoleic acid (data not shown).
We also compared the K+ current response to linoleic acid under two different holding potentials of –80 and –40 mV (Fig. 4). A similar reduction in the K+ current was observed with either holding potential (Fig. 4), which reconfirmed that linoleic acid significantly reduced the amplitude and accelerated the inactivation of the IK current.
In addition, this reduction in K+ current occurred immediately (within 2 min) after application of linoleic acid and reached the maximum decrement within 8–10 min. This effect was reversible within 20 min of the linoleic acid removal from the bath solution. The steady-state current of IK was measured at the end of the 200-msec depolarizing pulse from holding potential of –80 to +60 mV, and the measured values were used to form the time-response curve (Fig. 4C). The vehicle, which is H2O as linoleic acid solvent, diluted in bath solution at 1:1000, was used as a control and did not affect the K+ current value during 30 min recording time.
Effect of methyl linoleate on the voltage-gated K+ current
Methyl linoleate belongs to the long-chain FFAs group, with a structure similar to linoleic acid but has no binding affinity to GPR40 (4). The effect of methyl linoleate on the voltage-gated K+ current was examined to exclude the possibility of any influence on the K+ current via the nonreceptor-mediated way of FFAs. Local application of 10 μM methyl linoleate in bath solution for more than 10 min did not change the K+ current (Fig. 5Ab). On the same recorded cell, however, subsequent applications of 10 μM linoleic acid significantly reduced the K+ current (Fig. 5Ac). The statistical analysis of group data are shown in Fig. 5Ad. The GH3 cell line has no GPR40 mRNA expression and was used to exclude further nonreceptor-mediated effects of FFAs. Local application of 10 μM linoleic acid in bath solution for more than 10 min did not change the total K+ currents in GH3 cells (Fig. 5B)
Effect of GPR40-specific siRNA on the expression of GPR40 mRNA and K+ current response to FFAs in MIN6 cells
To further confirm that K+ current response to linoleic acid is mediated by GPR40, experiments were performed to inhibit the expression of GPR40 in MIN6 cells by incubating cells with siRNA for 48 h as reported (4). The MIN6 cell line retains similar characteristics to primary cultured -cells and shows a high transfection rate of GPR40-specific siRNA (4). Treatment with this siRNA significantly reduced GPR40 mRNA expression to less than half of the control level but had no effect on glucagon-like peptide-1-(7–36)-amide receptor (GLP-IR) mRNA expression (Fig. 6A). The decrement of K+ current response in MIN6 cells to linoleic acid was also significantly decreased by the siRNA treatment (Fig. 6B). The cell-permeable cAMP analog, 8-bromo-cAMP, induced a similar reduction of K+ current in MIN6 cells, and this reduction was not affected by the GPR40-specific siRNA treatment (Fig. 6C).
Involvement of protein kinases in the K+ current response to FFAs
Chelerythrine is a specific inhibitor of protein kinase C (PKC). Incubation with chelerythrine (1 μM) for 10 min did not change the K+ current or the K+ current response to linoleic acid (Fig. 7A). Rp-cAMP is a membrane permeable cAMP competitive antagonist, and H89 is a selective PKA inhibitor. Incubation with H89 (1 μM) for 10 min did not change the K+ current but abolished the K+ current response to linoleic acid (Fig. 7B). Incubation with Rp-cAMP (10 μM) for 10 min at room temperature did not change the voltage-gated K+ current (Fig. 7C). The K+ current response to linoleic acid was, however, totally abolished (Fig. 7C). To confirm whether the effect of FFAs on the voltage-gated K+ current was mediated through the cAMP-PKA system, adenyl cyclase stimulator forskolin and 8-bromo-cAMP were applied onto recorded -cells. Local application of 10 μM forskolin or 0.5 mM 8-bromo-cAMP in bath solution for more than 5 min induced a similar reduction in K+ current as that induced by linoleic acid (Fig. 7, D and E). Subsequent addition of 10 μM linoleic acid did not modify the K+ current further (Fig. 7, D and E).
cAMP assay in MIN6 cell line after treatment with FFAs
To confirm the K+ current response to linoleic acid depends on the cAMP-PKA system, intracellular cAMP levels have been measured in MIN6 mouse -cell line in different treatment conditions. The MIN6 cell line keeps similar characteristics to primary cultured -cells, and supply of cells is unlimited, compared with less than 200 islets from one rat, for the intracellular cAMP and secreted insulin assay. The size of every islet varies significantly, and total intracellular cAMP levels should be equally different between groups of islets. We therefore chose MIN6 cells to investigate the effect of linoleic acid on cAMP levels. Although linoleic acid alone did not increase significantly the cAMP levels in MIN6 cells, a significant enhancement of accumulated cAMP was observed when combined linoleic acid and IBMX (phosphodiesterase inhibitor) or forskolin (adenocyclase stimulator) were applied to cultured cells for 30 min (Fig. 8), and the increase in cAMP was significantly enhanced from IBMX- or forskolin-alone treatment. Preliminary experiments with an incubation time of 5 or 10 min showed a similar change of cAMP levels to linoleic acid stimulation (data not shown). A possible explanation for the discrepancy between linoleic acid alone and linoleic acid plus IBMX or forskolin is that linoleic acid may increase activities of both phosphodiesterase and adenocyclase to induce an increase in cAMP production and PKA activation without significant increase in accumulated cAMP in the cells.
Effect of linoleic acid on insulin secretion
The reduction in voltage-gated K+ current may enhance Ca2+ influx induced by membrane depolarization, which leads to insulin secretion from -cells. We finally examined the direct effect of linoleic acid on insulin secretion in cultured MIN6 cells. Because the K+ current response to linoleic acid was recorded in normal glucose condition (5 mM), the effect of linoleic acid on insulin secretion was tested in the same glucose concentration. When cells were incubated with different concentration of linoleic acid for 30 min, insulin secretion from MIN6 cells was increased dose dependently (Fig. 9A). When cells were incubated with linoleic acid, methyl linoleate, or the combination of both for 30 min, secreted insulin was measured in incubation medium. Methyl linoleate did not change insulin secretion, but linoleic acid significantly increased insulin secretion from cultured MIN6 cells (Fig. 9B).
Discussion
In this study, in vitro effects of the unsaturated FFA linoleic acid on the voltage-gated K+ currents in rat pancreatic -cells were examined. Local application of linoleic acid significantly reduced the amplitude of IK current and accelerated the inactivation of this current as demonstrated by nystatin-perforated whole-cell recording. This reduction in the K+ current is mediated by intracellular cAMP-PKA system, but not PKC system, through the GPR40 receptor.
Voltage-gated K+ channels (Kv) are among the main K+ channels in pancreatic -cells, and the physiological role of KV channels in -cells is to restore the cell membrane to a hyperpolarized state after a Ca2+-dependent action potential (6). The voltage-gated K+ current generated by Kv channels is composed of mainly two subtypes, a fast transient current, IA, and a slow inactivating delayed rectifier current, IK (21, 22). Both currents are reported to exist in pancreatic -cells, with IK being the predominant one (23, 24, 25). In this study, the voltage-gated K+ currents in rat pancreatic -cells were characterized on the basis of their electrophysiological properties. According to the previously reported biophysical characters, the IA current is elicited when a cell is depolarized from a holding potential of –80 mV but eliminated by a holding potential of –40 mV, whereas the IK is evoked when a cell is depolarized from a holding potential of either –80 or –40 mV (19). By using these two different holding potentials and then using a computerized subtraction program, it is suggested that the IA current is only a very small portion of the total voltage-gated K+ currents, which are mainly (about 95%) composed of the IK current (Fig. 2). By decreasing voltage-gated K+ currents in pancreatic -cells, an increase in the frequency and duration of action potential is expected, which then contributes to the elevation of [Ca2+]i and insulin secretion (26, 27, 28, 29). Reduction in the K+ current by linoleic acid therefore contributes to the insulin secretion by enhanced Ca2+-dependent exocytosis from the -cells. It should be emphasized that linoleic acid also evokes an increase in intracellular Ca2+ release through inositol 1, 4, 5-triphosphate-sensitive Ca2+ storage sites and activates the PKC system (30).
Glucose is the major modulator of -cell function, with even a small alteration in blood glucose concentration evoking large amounts of insulin secretion (30). FFAs are also important in maintaining -cell function as not only nutrients (30) but also critical factors to stimulate the insulin secretion from pancreatic -cells and keep a certain level of basal insulin in the plasma (31). The detailed mechanism underlying the short-term stimulatory effect of FFAs on insulin secretion is not clear. Previous studies have generally focused on the intracellular metabolism of FFAs and generation of lipid-derived molecules. FFAs are thought to enter cells across the plasma membrane and be activated by forming long chain acyl-CoA, which is considered to be an essential effector in signal transduction mechanisms in -cells (32). Acyl-CoA induces several intracellular effects, including stimulation of PKC, acylation of several proteins, modulation of protein trafficking, and direct activation of insulin exocytosis (32). GPR40 is a GPCR, and the specific endogenous ligands of this receptor have recently been demonstrated by several laboratories as long-chain FFAs (4, 5, 14). It is therefore proposed that FFAs stimulate insulin secretion by acting on the GPR40 receptor. High expression of GPR40 receptor is seen in several pancreatic -cell lines and primary cultured rat pancreatic islets in our experiment (Fig. 1), which is compatible with previous reports (4). We therefore tested the effect of linoleic acid in rat pancreatic -cells. We also illustrate that the methyl linoleate, which has a similar chemical structure to linoleic acid but no binding affinity to GPR40 (4), did not affect the voltage-gated K+ currents in rat -cells (Fig. 7). Furthermore, linoleic acid did not affect Kv currents in rat pituitary GH3 cells, which we demonstrate is without any GPR40 mRNA expression. Furthermore, siRNA experiments in MIN6 cells showed that the voltage-gated K+ current response to linoleic acid was impaired by the treatment of cells with a GPR40-specific siRNA, whereas the signal after GPR40, cAMP-induced reduction of K+ current was not affected by this siRNA treatment. These data strongly suggest that linoleic acid reduces Kv currents by activating GPR40.
FFAs increase [Ca2+]i through both Ca2+ influx via membrane Ca2+ channels, and Ca2+ release from intracellular Ca2+ storage sites, by acting on GPR40 and subsequent G protein-coupled signaling systems in pancreatic -cells (4). Opening of Ca2+ channels on the cell membrane is controlled by membrane potential, and the modification of cell electrophysiological properties changes the opening probability of Ca2+ channels. It has previously been hypothesized that long-chain FFAs may act on GPR40 to affect the activation of different ion channels on the -cell membrane, leading to the alteration of Ca2+ influx (29). In this study, the observed reduction of voltage-gated K+ currents in rat pancreatic -cells by linoleic acid may facilitate the Ca2+-influx through voltage-gated Ca2+ channels and thereafter contributes to insulin exocytosis. The supposed enhancement of the Ca2+-influx would be due to a prolonged repolarization phase of -cell action potentials, and the resultant rise in [Ca2+]i would enhance stimulation-induced insulin secretion. Detailed analysis of current traces with holding potentials of –80 and –40 mV indicates that linoleic acid not only decreased the amplitude of IK, but also changed the kinetics of this current by accelerating channel inactivation (Fig. 4). The time course of the response indicates that linoleic acid reduces the Kv currents in a few minutes, suggesting the involvement of an intracellular signal system employed by GPR40.
Kv channels belong to the six-transmembrane domain protein family of K+-selective channels (25, 33, 34), and several types of Kv channels have been identified in insulin-secreting cells (25, 33, 34). The Kv2.1 channel (biophysical characteristics of the IK channels for IK current) is reported as the main channel conducting the voltage-gated outward K+ currents in rat and mouse -cells (25). The C-terminal of Kv2.1 is involved in channel targeting (35), and the Kv2.1 -subunit C terminus contains two PKA-specific phosphorylation sites; modification of PKA leads to an alteration of currents through Kv2.1 channels recombinantly expressed in oocytes (36). In lymphocytes and cardiac myocytes, the cAMP/PKA-signaling pathway has also been implicated in the regulation of the Kv channels (37, 38). In addition, the GPCR-mediated inhibition of KV channel currents in rat pancreatic -cells is achieved by elevation of cAMP and activation of PKA (39). Recent reports suggest that FFAs increase intracellular Ca2+ and MAPK in MIN6 cells. The signaling pathway is coupled to the G protein subunit Gq/11 and partially to Gi proteins (4), leading to an activation of phospholipase C and hence activation of PKC and mobilization of Ca2+ from endoplasmic reticulum. In our current studies, the membrane permeable cAMP antagonist Rp-cAMP and the selective PKA inhibitor H89 were used to block the cAMP-PKA pathway, which eliminated the linoleic acid-induced reduction in voltage-gated K+ currents in rat -cells. In addition, the adenyl cyclase stimulator forskolin and cAMP analog 8-bromo-cAMP induced a similar reduction in the K+ current as that evoked by linoleic acid, which indicates that an increase in cAMP decreases the Kv currents in pancreatic -cells. The intracellular cAMP assay also showed a significantly enhanced increase in cAMP levels when linoleic acid was added with either forskolin or IBMX, in comparison with the addition of linoleic acid alone in MIN6 -cells. Chelerythrine, a specific PKC blocker, had no effect on the K+ current and its response to linoleic acid. These results suggest that FFAs may act on GPR40 to activate Gs protein, and through cAMP/PKA signaling pathway to reduce the Kv currents in -cells.
Conclusion
In conclusion, this report reveals that the long-chain unsaturated FFA, linoleic acid, reduces the voltage-gated IK current through the cAMP-PKA signaling pathway in pancreatic -cells. The GPR40 receptor, specific for long chain FFA, may be involved in the activation of the cAMP-PKA system. This reduction in the K+ conductance would prolong the membrane depolarization during action potential firing, leading to an increase in Ca2+ influx and insulin secretion from pancreatic -cells. This may serve as part of the molecular mechanism for FFAs to stimulate immediate insulin secretion.
Acknowledgments
We are grateful to Dr. P. Marley (Department of Pharmacology, Melbourne University) for his help on establishing cAMP assay methods and Dr. S. Hinuma (Frontier Research Laboratories, Takeda Pharmaceutical Co., Ibaraki-ken, Japan) for help on establishing siRNA method. We also thank D. Arnold and S. Panckridge for editorial help.
Footnotes
This work was supported by the Eli Lilly Endocrinology Program and Australian National Health and Medical Research Council.
First Published Online October 27, 2005
Abbreviations: 8-Bromo-cAMP, 8-bromoadenosine-cAMP; [Ca2+]i, intracellular free calcium concentration; FBS, fetal bovine serum; FFA, free fatty acid; GPCR, G protein-coupled receptor; GPR40, G protein-coupled receptor 40; H89, N-(2-(p-vromo-cinnamyl-amino)-ethyl)-5- iso-quinoline-sulfonamide; IBMX, 3-isobutyl-1-methylxanthine; IA, transient outward K+ current; IK, delayed rectifying K+ current; KRB, Krebs-Ringer bicarbonate; Kv, voltage-gated K+ channel; PKA, protein kinase A; PKC, protein kinase C; Rp-cAMP, adenosine-3',5'-cyclic phosphorothiolate-Rp-isomer; RT, reverse transcription; si, small interfering.
Accepted for publication October 19, 2005.
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Department of Physiology (D.D.F., Z.L.), XiangYa Medical School, Central-South University, Hunan 410018, China
Department of Food Production Science (S.R.), Shinshu University, Nagano-ken 399-4598, Japan
Abstract
Free fatty acids (FFAs), in addition to glucose, have been shown to stimulate insulin release through the G protein-coupled receptor (GPCR)40 receptor in pancreatic -cells. Intracellular free calcium concentration ([Ca2+]i) in -cells is elevated by FFAs, although the mechanism underlying the [Ca2+]i increase is still unknown. In this study, we investigated the action of linoleic acid on voltage-gated K+ currents. Nystatin-perforated recordings were performed on identified rat -cells. In the presence of nifedipine, tetrodotoxin, and tolbutamide, voltage-gated K+ currents were observed. The transient current represents less than 5%, whereas the delayed rectifier current comprises more than 95%, of the total K+ currents. A long-chain unsaturated FFA, linoleic acid (10 μM), reversibly decreased the amplitude of K+ currents (to less than 10%). This reduction was abolished by the cAMP/protein kinase A system inhibitors H89 (1 μM) and Rp-cAMP (10 μM) but was not affected by protein kinase C inhibitor. In addition, forskolin and 8'-bromo-cAMP induced a similar reduction in the K+ current as that evoked by linoleic acid. Insulin secretion and cAMP accumulation in -cells were also increased by linoleic acid. Methyl linoleate, which has a similar structure to linoleic acid but no binding affinity to GPR40, did not change K+ currents. Treatment of cultured cells with GPR40-specific small interfering RNA significantly reduced the decrease in K+ current induced by linoleic acid, whereas the cAMP-induced reduction of K+ current was not affected. We conclude that linoleic acid reduces the voltage-gated K+ current in rat -cells through GPR40 and the cAMP-protein kinase A system, leading to an increase in [Ca2+]i and insulin secretion.
Introduction
IT IS APPARENT that excessive fat tissue or obese conditions facilitate the progress of type 2 diabetes and may be responsible for the dramatic increase in the occurrence of diabetes in recent years. Abnormal metabolism of carbohydrate and lipid has been found in type 2 diabetes with reduced efficacy of insulin (insulin resistance) in muscle, liver, and other tissues (1, 2). Also, the release of insulin from pancreatic -cells in response to increased blood glucose is elevated in the early stage of diabetes to compensate insulin resistance. Free fatty acids (FFAs) are mainly released from adipose tissue as an energy source, and the acute elevation of plasma FFAs is necessary for insulin secretion (3). The detailed mechanism for such stimulation is, however, not clearly understood.
Recent reports provide evidence that long-chain FFAs are specific ligands of an orphan G protein-coupled receptor (GPCR), GPR40 (4, 5). GPR40 is abundantly expressed in pancreatic -cells and insulin-secreting cell lines and its structure is highly conserved. Functionally, FFAs increase insulin release and amplify glucose-stimulated insulin secretion through an increase in intracellular free calcium concentration ([Ca2+]i), via both Ca2+ influx through Ca2+ channels and Ca2+ release from intracellular storage sites in cultured -cell lines (4).
Regulation of insulin secretion by glucose involves the coordinated control of ion channels in the -cell membrane (6). The initial response of -cells to glucose is closure of the ATP-sensitive K+ channels. This leads to membrane depolarization followed by the activation of Ca2+ influx through voltage-gated Ca2+ channels (6). In addition, voltage-gated outward K+ currents have been detected in -cells and are demonstrated to mediate membrane potential repolarization and limit Ca2+ influx and insulin secretion (7, 8, 9, 10). It is not yet established whether voltage-gated K+ channels are directly affected by glucose and FFAs.
In the present study, we characterized voltage-gated K+ currents and demonstrated the effect of the unsaturated long-chain FFA, linoleic acid, on K+ currents in primary cultured rat pancreatic -cells. We further elucidated possible receptor and signaling systems employed by linoleic acid in the MIN6 -cell line using small interfering (si) RNA and biochemical assays. Our data suggest that such an effect is mediated by GPR40 and the cAMP-protein kinase A (PKA) signal transduction system.
Materials and Methods
Chemical reagents
Linoleic acid and methyl linoleate were purchased from Sigma (St. Louis, MO). Histopaque-1077, protease (Dispase), and collagenase type V were also obtained from Sigma. RPMI 1640, DMEM, HEPES, and carbohydrate solution was purchased from Thermo Electron (Melbourne, Victoria, Australia). Nifedipine, tolbutamide, nystatin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, 8'-bromo-cAMP and all general salts for recording solutions and molecular biological studies were purchased from Sigma. Tetrodotoxin was purchased from Alomone Laboratories (Jerusalem, Israel). Chelerythrine chloride, adenosine-3',5'-cyclic phosphorothiolate-RP (Rp-cAMP), and N-(2-(p-vromo-cinnamyl-amino)-ethyl)-5-iso-quinoline-sulfonamide (H89) were purchased from CalBiochem (Alexandria, New South Wales, Australia).
Cell culture and preparation
Pancreatic islets were isolated mostly from male Sprague Dawley rats, with an additional few from male Wistar rats (8–10 wk). We compared -cells from two strains and did not find any differences in the K+ current and the K+ current response to linoleic acid. After the pancreas was injected with 10 ml collagenase solution (1.5 mg/ml collagenase in Hank’s solution) through the common bile duct, the tissue was incubated at 37 C for 30 min. The digested pancreas was then washed, filtered through 355 μm mesh, and resuspended in cold RPMI 1640 medium. Islets were isolated using a density gradient created by different concentrations of histopaque-1077 in RPMI 1640 medium after centrifugation (11). Purified islets were further dispersed into single cells by digestion with 1 mg/ml Dispase for 10 min at room temperature. Cells were then seeded into 35-mm dishes in RPMI 1640 medium supplemented by 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured in a humidified atmosphere containing 5% CO2 at 37 C. The culture medium was replenished every 2 d, and all electrophysiological recordings were performed on the rat pancreatic -cells cultured for 2–10 d. Pancreatic -cells accounted for over 70% of total cultured cells and were identified by their significantly larger size. In addition, membrane depolarization by 15 mM glucose was tested in more than 20 cells in early establishment of recording system on primary cultured islet cells to confirm that the recorded cells were -cells. The K+ current and response to linoleic acid in the cells cultured over different lengths of time were carefully evaluated, and no difference was found. The MIN6 cell line (Walter and Eliza Institute, Melbourne, Australia) is a mouse pancreatic -cell line that retains the morphological characteristics of the pancreatic -cells (12). These cells were routinely cultured in 25 mM glucose DMEM + 10% FBS + 1% penicillin/streptomycin medium.
RNA isolation and RT-PCR
The total RNA from cells (1 x 106cells/dish) and rat islets (300 islets/dish) cultured in 35-mm petri dishes was extracted by established Trizol method (13). The total RNA from mouse heart and kidney tissue is from another laboratory in the institute. One microgram of total RNA was then reverse transcribed to cDNA in a 20-μl reverse transcription (RT) reaction system containing random primers and avian myeloblastosis virus reverse transcriptase. The RT reactions were carried out at 46 C for 2 h. One microgram of the RT reaction product was used for subsequent PCR amplification. Primer for rat GPR40 was designed according to the rat cDNA templates with an expected size of 79 bp. The set of primers for the amplification of the GPR40 was 5'-CCTATAATGCTTCCAATGTGGCTAGT-3' (forward) and 5'-CCTGTGATGAGCCCCAACTT-3' (reverse) (14). We used a denaturation step at 95 C for 30 sec, an annealing step at 60 C for 30 sec, and an extension step at 72 C for 30 sec for a total of 25 cycles, followed by an additional extension step at 72 C for 5 min. Detection of PCR amplification products was carried out by electrophoresis (1.4% agarose gel containing ethidium bromide).
Electrophysiological recording
On the day of recording, culture medium was discarded and cells were incubated in bath solution for 10–20 min before recording. Transmembrane currents were recorded using the nystatin-perforated method in whole cell recording configuration (15, 16). Electrodes were pulled by a Sutter P-87 microelectrode puller (Sutter Equipment Co., Navato, CA) from borosilicate micropipettes with inner filament. After fire polishing, electrodes had an initial tip resistance of 5–8 M. All recordings were made using the Axopatch 200 amplifier and pClamp7 software (Axon Instruments, Foster City, CA).
The standard bath solution was composed of the following (millimoles): 140 NaCl, 5 KCl, 2.5 CaCl2, 0.5 MgCl2, 5 glucose, and 10 HEPES (pH 7.4) and osmolarity of 310 mOsm. The pipette solution contained (millimoles): 55 KCl, 75 K2SO4, 8 MgSO4, and 10 HEPES (pH7.4) and osmolarity of 300 mOsm. The electrode was backfilled with this solution containing nystatin (200 μg/ml in 0.3% dimethyl sulfoxide). After obtaining a high-resistance seal, the holding potential was set to –80 mV and voltage pulses (30 mV, 200 msec) were delivered periodically to monitor the appearance of a membrane capacitance transient current, which usually occurred within 3–5 min under the experimental conditions, indicating formation of a low-resistance access to the cell. Typically the series resistance reached a steady level less than 35 M within 5 min of making a seal. Input resistance of cells was higher than 10 G under our recording conditions. Whole-cell capacitance (30 cells, 8 ± 0.42 pF) and series resistance were compensated, and leak current was routinely subtracted using the option offered by Clampex program. Isolation of voltage-gated K+ currents involved the exclusion of Ca2+ (1 μM nifedipine) and Na+ (1 μM tetrodotoxin) and ATP-sensitive K+ (0.1 mM tolbutamide) currents by adding the specific blockers of these channels into the bath solution before the recording.
Original cell culture dishes were placed on the stage of an inverted microscope (Olympus, Tokyo, Japan) and cells were continuously perfused using gravity-flow at a rate of 1 ml/min. Tested FFAs and intracellular signaling stimulators were applied to the same gravity system by switching solution reservoirs. Specific intracellular signaling inhibitors were applied by hand carefully with pipette and leave for more than 10 min without perfusion to reach calculated final concentration as indicated in Results section before starting perfusion of FFAs in presence of same concentration of inhibitors onto the recorded cell. Control studies were performed by applying vehicle instead of the drugs. Because -cells were reported to be 3-fold larger than -cells (17), only cells with a diameter larger than 10 μm and well-preserved granulations were used for the recording. Recordings were low-pass filtered at 5 kHz and stored on the hard disk of an IBM-compatible computer (IBM, White Plains, NY). The value of currents was measured separately around 30 msec and 200 msec of each evoked current trace for peak (total K+ current) or steady-state [delayed rectifying K+ current (IK) current only] current during 200-msec depolarizing pulses. All experiments were performed at room temperature (20–22 C).
Intracellular cAMP and insulin secretion
MIN6 insulinoma cells (passage 28–35) were cultured in 25 mM glucose DMEM with 10% FBS, and 1% penicillin/streptomycin at 37 C and 5% CO2. Two days before cAMP sample collection and insulin secretion experiments, cells were trypsinized and plated in 24-well plates at 5 x 105 cells/well. On the day of experiment, cells were washed and preincubated in Krebs-Ringer bicarbonate (KRB) buffer for 1 h.
For cAMP measurement, cells were extracted in 1 ml of acid ethanol on top of dry ice, and extracts were dried down in a vacuum centrifuge (Speed Vac, Savant, Hicksville, NY) and stored at –20 C until assayed. cAMP in the extracts was reconstituted and measured by a sensitive and specific RIA (18).
Insulin secretion experiments were performed in KRB buffer. The cells were treated for 90 min with FFAs dissolved in KRB. The amount of insulin in the culture supernatant was determined with an ELISA kit (Diagnostic Systems Laboratories, Webster, TX).
siRNA treatment procedure in cultured MIN6 cells
RNA oligonucleotides were designed targeting AA(N19) sequences in the open reading frame of mRNA encoding mouse GPR40 as described by Itoh et al. (4). Selected siRNA target sequences were also submitted to BLAST searches against other mouse genome sequences to ensure target specificity. The following siRNA (target sequences 5'-CGCCAGUUGUGACAUUCUUdTdT-3' and 5'-AAGAAUGUCACAACUGGCGdTdT-3') was the most potent in silencing mouse GPR40-GFP proteins expressed in CHO cells (4) and was therefore used in this experiment. Scrambled siRNA duplex was used as a control (4). MIN6 cells were plated in 35-mm dishes for 2 d before transfection. Transfection of siRNA was accomplished with HVJ envelope vector kit GenomONE (Ishihara Sangyo Kaisha, Tokyo, Japan) according to the manufacturer’s instructions. Cells were incubated at 37 C in a CO2 incubator, and the culture medium was replaced 24 h after transfection. Two days after transfection, MIN6 cells were collected for RNA isolation and the GPR40 RT-PCR experiment as well as electrophysiological recording.
Statistical analysis
pClamp software (version 8.2; Axon Instruments) was used to acquire data during the recording and analyze the patch-clamp data afterward. Student’s paired t test was used as appropriate to evaluate the statistical significance of differences between treatment and control group means, and the effect was considered to be significant at the level of less than 5%. Group data represent at least four recordings under the same experimental conditions and are expressed as mean ± SEM in Results.
Results
Identification of GPR40 mRNA expression in pancreatic islets and -cells
GPR40 mRNA expression was determined in pancreatic islets and -cells by RT-PCR. Rat pancreatic islets, Min6, and INS-1 cell lines have high levels of expression of GPR40 mRNA, whereas the expression of GPR40 mRNA was detected in mouse heart tissue but undetectable under the same PCR conditions in mouse kidney tissue and GH3 rat pituitary cell line, which were used as negative controls (Fig. 1). The size of amplified fragments of the receptor was 79 bp.
Characterization of the voltage-gated K+ currents
Voltage-gated K+ currents are mainly composed of delayed IK and transient outward (IA) currents. According to the biophysical characters, currents are evoked when the cell is depolarized from different holding potentials of –40 or –80 mV, respectively. Kinetically after activation, IA is rapidly inactivated, whereas IK is noninactivated or slow inactivated. To characterize the two subtypes of voltage-gated K+ currents, depolarizing steps from two holding potentials of –80 mV (Fig. 2A) and –40 mV (Fig. 2B) were applied, in the presence of the Ca2+, Na+ and ATP-sensitive K+ channel blockers (see Materials and Methods). According to voltage-dependent inactivation properties (19), the current generated from a holding potential of –80 mV contains both IA and IK as total currents (Fig. 2A), whereas the current evoked from a holding potential of –40 mV contains only the IK current (Fig. 2B). Subtraction of currents recorded with the holding potential of –40 mV from total currents with the holding potential of –80 mV using pClamp software shows a very small portion of currents left (Fig. 2C), which represents the IA current. It is obvious that IA is very small and almost undetectable in this recording (less than 5% of total K+ current in all cells recorded). The IA current therefore represents only a minor proportion of the total voltage-gated K+ currents in this recording condition in primary cultured rat -cells, whereas the major part is IK current.
Effect of FFAs on the voltage-gated K+ current
Depolarizing test pulses from a holding potential of –80 mV elicited the outward K+ current (Fig. 3Aa), mainly IK current. Local application of 10 μM linoleic acid [the dose chosen according to published data on insulin secretion (4)] in bath solution induced a significant decrease in the amplitude of the IK current. Levels of FFAs in plasma are 0.2–1.7 mM, with 99% of these FFAs bound tightly with serum albumin (4). The concentration of unbound FFAs is in the range of 0.01–10 μM. In addition, it has been reported that unbound FFAs act on GPR40 to release insulin and the effective dose was 10 μM (4). In this context, the concentration of linoleic acid used (10 μM) in this report is physiologically relevant. Because the current now inactivated rapidly, it seems that either linoleic acid accelerated the inactivation speed of the current or the induced reduction in the current depended on the open/close gating statues of the IK channel as reported previously (20) (Fig. 3Ab). After removal of linoleic acid for 10–20 min, the K+ current recovered to the control level (Fig. 3Ac). The current was measured at the peak value (30 msec) and end (200 msec) of pulse to obtain the current-voltage relationship for both time points (Fig. 3B). From a group of eight cells, such a reduction in K+ current by linoleic acid was statistically significant at both time points (P < 0.01, Fig. 3C). We also examined oleic acid and found a similar effect on the K+ current, although the reduction was not as remarkable as that reduced by linoleic acid (data not shown).
We also compared the K+ current response to linoleic acid under two different holding potentials of –80 and –40 mV (Fig. 4). A similar reduction in the K+ current was observed with either holding potential (Fig. 4), which reconfirmed that linoleic acid significantly reduced the amplitude and accelerated the inactivation of the IK current.
In addition, this reduction in K+ current occurred immediately (within 2 min) after application of linoleic acid and reached the maximum decrement within 8–10 min. This effect was reversible within 20 min of the linoleic acid removal from the bath solution. The steady-state current of IK was measured at the end of the 200-msec depolarizing pulse from holding potential of –80 to +60 mV, and the measured values were used to form the time-response curve (Fig. 4C). The vehicle, which is H2O as linoleic acid solvent, diluted in bath solution at 1:1000, was used as a control and did not affect the K+ current value during 30 min recording time.
Effect of methyl linoleate on the voltage-gated K+ current
Methyl linoleate belongs to the long-chain FFAs group, with a structure similar to linoleic acid but has no binding affinity to GPR40 (4). The effect of methyl linoleate on the voltage-gated K+ current was examined to exclude the possibility of any influence on the K+ current via the nonreceptor-mediated way of FFAs. Local application of 10 μM methyl linoleate in bath solution for more than 10 min did not change the K+ current (Fig. 5Ab). On the same recorded cell, however, subsequent applications of 10 μM linoleic acid significantly reduced the K+ current (Fig. 5Ac). The statistical analysis of group data are shown in Fig. 5Ad. The GH3 cell line has no GPR40 mRNA expression and was used to exclude further nonreceptor-mediated effects of FFAs. Local application of 10 μM linoleic acid in bath solution for more than 10 min did not change the total K+ currents in GH3 cells (Fig. 5B)
Effect of GPR40-specific siRNA on the expression of GPR40 mRNA and K+ current response to FFAs in MIN6 cells
To further confirm that K+ current response to linoleic acid is mediated by GPR40, experiments were performed to inhibit the expression of GPR40 in MIN6 cells by incubating cells with siRNA for 48 h as reported (4). The MIN6 cell line retains similar characteristics to primary cultured -cells and shows a high transfection rate of GPR40-specific siRNA (4). Treatment with this siRNA significantly reduced GPR40 mRNA expression to less than half of the control level but had no effect on glucagon-like peptide-1-(7–36)-amide receptor (GLP-IR) mRNA expression (Fig. 6A). The decrement of K+ current response in MIN6 cells to linoleic acid was also significantly decreased by the siRNA treatment (Fig. 6B). The cell-permeable cAMP analog, 8-bromo-cAMP, induced a similar reduction of K+ current in MIN6 cells, and this reduction was not affected by the GPR40-specific siRNA treatment (Fig. 6C).
Involvement of protein kinases in the K+ current response to FFAs
Chelerythrine is a specific inhibitor of protein kinase C (PKC). Incubation with chelerythrine (1 μM) for 10 min did not change the K+ current or the K+ current response to linoleic acid (Fig. 7A). Rp-cAMP is a membrane permeable cAMP competitive antagonist, and H89 is a selective PKA inhibitor. Incubation with H89 (1 μM) for 10 min did not change the K+ current but abolished the K+ current response to linoleic acid (Fig. 7B). Incubation with Rp-cAMP (10 μM) for 10 min at room temperature did not change the voltage-gated K+ current (Fig. 7C). The K+ current response to linoleic acid was, however, totally abolished (Fig. 7C). To confirm whether the effect of FFAs on the voltage-gated K+ current was mediated through the cAMP-PKA system, adenyl cyclase stimulator forskolin and 8-bromo-cAMP were applied onto recorded -cells. Local application of 10 μM forskolin or 0.5 mM 8-bromo-cAMP in bath solution for more than 5 min induced a similar reduction in K+ current as that induced by linoleic acid (Fig. 7, D and E). Subsequent addition of 10 μM linoleic acid did not modify the K+ current further (Fig. 7, D and E).
cAMP assay in MIN6 cell line after treatment with FFAs
To confirm the K+ current response to linoleic acid depends on the cAMP-PKA system, intracellular cAMP levels have been measured in MIN6 mouse -cell line in different treatment conditions. The MIN6 cell line keeps similar characteristics to primary cultured -cells, and supply of cells is unlimited, compared with less than 200 islets from one rat, for the intracellular cAMP and secreted insulin assay. The size of every islet varies significantly, and total intracellular cAMP levels should be equally different between groups of islets. We therefore chose MIN6 cells to investigate the effect of linoleic acid on cAMP levels. Although linoleic acid alone did not increase significantly the cAMP levels in MIN6 cells, a significant enhancement of accumulated cAMP was observed when combined linoleic acid and IBMX (phosphodiesterase inhibitor) or forskolin (adenocyclase stimulator) were applied to cultured cells for 30 min (Fig. 8), and the increase in cAMP was significantly enhanced from IBMX- or forskolin-alone treatment. Preliminary experiments with an incubation time of 5 or 10 min showed a similar change of cAMP levels to linoleic acid stimulation (data not shown). A possible explanation for the discrepancy between linoleic acid alone and linoleic acid plus IBMX or forskolin is that linoleic acid may increase activities of both phosphodiesterase and adenocyclase to induce an increase in cAMP production and PKA activation without significant increase in accumulated cAMP in the cells.
Effect of linoleic acid on insulin secretion
The reduction in voltage-gated K+ current may enhance Ca2+ influx induced by membrane depolarization, which leads to insulin secretion from -cells. We finally examined the direct effect of linoleic acid on insulin secretion in cultured MIN6 cells. Because the K+ current response to linoleic acid was recorded in normal glucose condition (5 mM), the effect of linoleic acid on insulin secretion was tested in the same glucose concentration. When cells were incubated with different concentration of linoleic acid for 30 min, insulin secretion from MIN6 cells was increased dose dependently (Fig. 9A). When cells were incubated with linoleic acid, methyl linoleate, or the combination of both for 30 min, secreted insulin was measured in incubation medium. Methyl linoleate did not change insulin secretion, but linoleic acid significantly increased insulin secretion from cultured MIN6 cells (Fig. 9B).
Discussion
In this study, in vitro effects of the unsaturated FFA linoleic acid on the voltage-gated K+ currents in rat pancreatic -cells were examined. Local application of linoleic acid significantly reduced the amplitude of IK current and accelerated the inactivation of this current as demonstrated by nystatin-perforated whole-cell recording. This reduction in the K+ current is mediated by intracellular cAMP-PKA system, but not PKC system, through the GPR40 receptor.
Voltage-gated K+ channels (Kv) are among the main K+ channels in pancreatic -cells, and the physiological role of KV channels in -cells is to restore the cell membrane to a hyperpolarized state after a Ca2+-dependent action potential (6). The voltage-gated K+ current generated by Kv channels is composed of mainly two subtypes, a fast transient current, IA, and a slow inactivating delayed rectifier current, IK (21, 22). Both currents are reported to exist in pancreatic -cells, with IK being the predominant one (23, 24, 25). In this study, the voltage-gated K+ currents in rat pancreatic -cells were characterized on the basis of their electrophysiological properties. According to the previously reported biophysical characters, the IA current is elicited when a cell is depolarized from a holding potential of –80 mV but eliminated by a holding potential of –40 mV, whereas the IK is evoked when a cell is depolarized from a holding potential of either –80 or –40 mV (19). By using these two different holding potentials and then using a computerized subtraction program, it is suggested that the IA current is only a very small portion of the total voltage-gated K+ currents, which are mainly (about 95%) composed of the IK current (Fig. 2). By decreasing voltage-gated K+ currents in pancreatic -cells, an increase in the frequency and duration of action potential is expected, which then contributes to the elevation of [Ca2+]i and insulin secretion (26, 27, 28, 29). Reduction in the K+ current by linoleic acid therefore contributes to the insulin secretion by enhanced Ca2+-dependent exocytosis from the -cells. It should be emphasized that linoleic acid also evokes an increase in intracellular Ca2+ release through inositol 1, 4, 5-triphosphate-sensitive Ca2+ storage sites and activates the PKC system (30).
Glucose is the major modulator of -cell function, with even a small alteration in blood glucose concentration evoking large amounts of insulin secretion (30). FFAs are also important in maintaining -cell function as not only nutrients (30) but also critical factors to stimulate the insulin secretion from pancreatic -cells and keep a certain level of basal insulin in the plasma (31). The detailed mechanism underlying the short-term stimulatory effect of FFAs on insulin secretion is not clear. Previous studies have generally focused on the intracellular metabolism of FFAs and generation of lipid-derived molecules. FFAs are thought to enter cells across the plasma membrane and be activated by forming long chain acyl-CoA, which is considered to be an essential effector in signal transduction mechanisms in -cells (32). Acyl-CoA induces several intracellular effects, including stimulation of PKC, acylation of several proteins, modulation of protein trafficking, and direct activation of insulin exocytosis (32). GPR40 is a GPCR, and the specific endogenous ligands of this receptor have recently been demonstrated by several laboratories as long-chain FFAs (4, 5, 14). It is therefore proposed that FFAs stimulate insulin secretion by acting on the GPR40 receptor. High expression of GPR40 receptor is seen in several pancreatic -cell lines and primary cultured rat pancreatic islets in our experiment (Fig. 1), which is compatible with previous reports (4). We therefore tested the effect of linoleic acid in rat pancreatic -cells. We also illustrate that the methyl linoleate, which has a similar chemical structure to linoleic acid but no binding affinity to GPR40 (4), did not affect the voltage-gated K+ currents in rat -cells (Fig. 7). Furthermore, linoleic acid did not affect Kv currents in rat pituitary GH3 cells, which we demonstrate is without any GPR40 mRNA expression. Furthermore, siRNA experiments in MIN6 cells showed that the voltage-gated K+ current response to linoleic acid was impaired by the treatment of cells with a GPR40-specific siRNA, whereas the signal after GPR40, cAMP-induced reduction of K+ current was not affected by this siRNA treatment. These data strongly suggest that linoleic acid reduces Kv currents by activating GPR40.
FFAs increase [Ca2+]i through both Ca2+ influx via membrane Ca2+ channels, and Ca2+ release from intracellular Ca2+ storage sites, by acting on GPR40 and subsequent G protein-coupled signaling systems in pancreatic -cells (4). Opening of Ca2+ channels on the cell membrane is controlled by membrane potential, and the modification of cell electrophysiological properties changes the opening probability of Ca2+ channels. It has previously been hypothesized that long-chain FFAs may act on GPR40 to affect the activation of different ion channels on the -cell membrane, leading to the alteration of Ca2+ influx (29). In this study, the observed reduction of voltage-gated K+ currents in rat pancreatic -cells by linoleic acid may facilitate the Ca2+-influx through voltage-gated Ca2+ channels and thereafter contributes to insulin exocytosis. The supposed enhancement of the Ca2+-influx would be due to a prolonged repolarization phase of -cell action potentials, and the resultant rise in [Ca2+]i would enhance stimulation-induced insulin secretion. Detailed analysis of current traces with holding potentials of –80 and –40 mV indicates that linoleic acid not only decreased the amplitude of IK, but also changed the kinetics of this current by accelerating channel inactivation (Fig. 4). The time course of the response indicates that linoleic acid reduces the Kv currents in a few minutes, suggesting the involvement of an intracellular signal system employed by GPR40.
Kv channels belong to the six-transmembrane domain protein family of K+-selective channels (25, 33, 34), and several types of Kv channels have been identified in insulin-secreting cells (25, 33, 34). The Kv2.1 channel (biophysical characteristics of the IK channels for IK current) is reported as the main channel conducting the voltage-gated outward K+ currents in rat and mouse -cells (25). The C-terminal of Kv2.1 is involved in channel targeting (35), and the Kv2.1 -subunit C terminus contains two PKA-specific phosphorylation sites; modification of PKA leads to an alteration of currents through Kv2.1 channels recombinantly expressed in oocytes (36). In lymphocytes and cardiac myocytes, the cAMP/PKA-signaling pathway has also been implicated in the regulation of the Kv channels (37, 38). In addition, the GPCR-mediated inhibition of KV channel currents in rat pancreatic -cells is achieved by elevation of cAMP and activation of PKA (39). Recent reports suggest that FFAs increase intracellular Ca2+ and MAPK in MIN6 cells. The signaling pathway is coupled to the G protein subunit Gq/11 and partially to Gi proteins (4), leading to an activation of phospholipase C and hence activation of PKC and mobilization of Ca2+ from endoplasmic reticulum. In our current studies, the membrane permeable cAMP antagonist Rp-cAMP and the selective PKA inhibitor H89 were used to block the cAMP-PKA pathway, which eliminated the linoleic acid-induced reduction in voltage-gated K+ currents in rat -cells. In addition, the adenyl cyclase stimulator forskolin and cAMP analog 8-bromo-cAMP induced a similar reduction in the K+ current as that evoked by linoleic acid, which indicates that an increase in cAMP decreases the Kv currents in pancreatic -cells. The intracellular cAMP assay also showed a significantly enhanced increase in cAMP levels when linoleic acid was added with either forskolin or IBMX, in comparison with the addition of linoleic acid alone in MIN6 -cells. Chelerythrine, a specific PKC blocker, had no effect on the K+ current and its response to linoleic acid. These results suggest that FFAs may act on GPR40 to activate Gs protein, and through cAMP/PKA signaling pathway to reduce the Kv currents in -cells.
Conclusion
In conclusion, this report reveals that the long-chain unsaturated FFA, linoleic acid, reduces the voltage-gated IK current through the cAMP-PKA signaling pathway in pancreatic -cells. The GPR40 receptor, specific for long chain FFA, may be involved in the activation of the cAMP-PKA system. This reduction in the K+ conductance would prolong the membrane depolarization during action potential firing, leading to an increase in Ca2+ influx and insulin secretion from pancreatic -cells. This may serve as part of the molecular mechanism for FFAs to stimulate immediate insulin secretion.
Acknowledgments
We are grateful to Dr. P. Marley (Department of Pharmacology, Melbourne University) for his help on establishing cAMP assay methods and Dr. S. Hinuma (Frontier Research Laboratories, Takeda Pharmaceutical Co., Ibaraki-ken, Japan) for help on establishing siRNA method. We also thank D. Arnold and S. Panckridge for editorial help.
Footnotes
This work was supported by the Eli Lilly Endocrinology Program and Australian National Health and Medical Research Council.
First Published Online October 27, 2005
Abbreviations: 8-Bromo-cAMP, 8-bromoadenosine-cAMP; [Ca2+]i, intracellular free calcium concentration; FBS, fetal bovine serum; FFA, free fatty acid; GPCR, G protein-coupled receptor; GPR40, G protein-coupled receptor 40; H89, N-(2-(p-vromo-cinnamyl-amino)-ethyl)-5- iso-quinoline-sulfonamide; IBMX, 3-isobutyl-1-methylxanthine; IA, transient outward K+ current; IK, delayed rectifying K+ current; KRB, Krebs-Ringer bicarbonate; Kv, voltage-gated K+ channel; PKA, protein kinase A; PKC, protein kinase C; Rp-cAMP, adenosine-3',5'-cyclic phosphorothiolate-Rp-isomer; RT, reverse transcription; si, small interfering.
Accepted for publication October 19, 2005.
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