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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on June 16, 2004; DOI: 10.1124/jpet.104.067249

0022-3565/04/3103-1273-1280$20.00
JPET 310:1273-1280, 2004
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CELLULAR AND MOLECULAR

Fenofibrate, Troglitazone, and 15-Deoxy-{Delta}12,14-prostaglandin J2 Close KATP Channels and Induce Insulin Secretion

Kenju Shimomura, Hiroyuki Shimizu, Mizuho Ikeda, Shuichi Okada, Masafumi Kakei, Shigeji Matsumoto, and Masatomo Mori

Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan (K.S., H.S., S.O., M.M.); Department of Physiology, Nippon Dental University School of Dentistry at Tokyo, Tokyo, Japan (M.I., S.M.); and Department of Geriatric Medicine, Akita University School of Medicine, Akita, Japan (M.K.)

Received February 23, 2004; accepted June 15, 2004.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
It is known that peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) ligands stimulate acute-phase insulin secretion with a rapid Ca2+ influx into pancreatic {beta}-cells, but the precise mechanisms are not clear. The effects of PPAR-{alpha} ligands on pancreatic {beta}-cells also remain unclear. We investigated the effects of PPAR-{alpha} ligands (fenofibrate and fenofibric acid), a PPAR-{gamma} ligand (troglitazone), and an endogenous ligand of PPAR-{gamma} [15-deoxy-{Delta}12,14-prostaglandin J2 (15-deoxy-{Delta}12,14-PGJ2)] on KATP channel activity in clonal hamster insulinoma cell line, HIT-T15 cells. As assessed by whole-cell patch clamp, fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 reduced the KATP channel currents, and inhibition continued after washout of these agents. The concentration-response curves of fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 showed half-maximal inhibition of KATP channel currents (IC50) at 3.26, 94, 2.1, and 7.3 µmol/l, respectively. Fenofibrate (≥ 10-6 mol/l), 15-deoxy-{Delta}12,14-PGJ2 (≥ 5 x 10-5 mol/l), and troglitazone (≥ 10-6 mol/l) inhibited [3H]glibenclamide binding, but fenofibric acid did not. In addition, fenofibrate (≥ 10-6 mol/l), fenofibric acid (10-4 mol/l), troglitazone (10-4 mol/l), and 15-deoxy-{Delta}12,14-PGJ2 (≥ 10-5 mol/l) increased insulin secretion from HIT-T15 when applied for 10 min. Our data suggest that PPAR-{alpha} and -{gamma} ligands interact directly with the {beta}-cell membrane and stimulate insulin secretion.

Insulin secretion is stimulated by the closure of the ATP-sensitive potassium (KATP) channel of the {beta}-cell membrane (Cook and Hales, 1984Go; Rorsman and Trube, 1985Go; Sturgess et al., 1985Go; Ashcroft and Gribble, 1999Go). The KATP channel plays a major role in regulating the membrane potential of {beta}-cells because it is able to respond to the metabolic state of the cell. The reduction in membrane K+ permeability by closure of the KATP channel depolarizes the {beta}-cell membrane. As a consequence of the change in membrane potential to more positive potentials, the voltage-dependent Ca2+ channel opens to allow Ca2+ influx and stimulates insulin release (Rorsman, 1997Go; Ashcroft and Gribble, 1999Go). Sulfonylureas, such as tolbutamide and glibenclamide, stimulate insulin secretion by interacting directly with the KATP channel (Sturgess et al., 1985Go; Edwards and Weston, 1993Go). KATP channels in pancreatic {beta}-cells are composed of an inwardly rectifying Kir6.2 channel subunit, which forms the channel pore, and the sulfonylurea receptor (SUR) 1 subunit, which acts as a regulatory subunit (Clement et al., 1997Go; Inagaki et al., 1997Go; Shyng and Nichols, 1997Go; Ashcroft and Gribble, 1999Go). Sulfonylureas are known to bind directly to SUR1 to inhibit the KATP channel (Sturgess et al., 1985Go; Edwards and Weston, 1993Go; Ashcroft and Gribble, 1999Go).

On the other hand, intracellular nuclear receptors and peroxisome proliferator-activated receptors (PPARs) have evident effects on protein synthesis (Braissant et al., 1996Go). PPAR subtypes {alpha}, {gamma}, and {delta} show distinctive tissue distributions and are associated with selective ligands (Braissant et al., 1996Go). Once activated by ligands, PPARs heterodimerize with the retinoic X receptor and alter the transcription of target genes after binding to response elements, consisting of a direct repeat of the nuclear receptor DNA recognition motif spaced by one nucleotide. PPAR-{alpha} is known to regulate fatty acid metabolism by controlling its oxidation, and PPAR-{gamma} is known to involve glucose homeostasis and adipocyte proliferation (Lemberger et al., 1996Go; Corton et al., 2000Go). Fenofibrate is a PPAR-{alpha} ligand and clinically used for the treatment of hyperlipidemia. Fenofibrate is rapidly converted into fenofibric acid in the liver and plasma. It is assumed that the fenofibric acid is the pharmacologically relevant form (Caldwell, 1989Go). On the other hand, troglitazone is a PPAR-{gamma} ligand, and 15-deoxy-{Delta}12,14-PGJ2 is known to be an endogenous ligand of PPAR-{gamma} (Kliewer et al., 1995Go).

We have previously demonstrated that the PPAR-{gamma} ligands troglitazone and pioglitazone both stimulate acute-phase insulin secretion with a rapid increase of cytoplasmic Ca2+ but inhibit chronic-phase insulin secretion in a Ca2+-dependent manner (Ohtani et al., 1996Go, 1998Go). The removal of extracellular Ca2+ abolished acute-phase insulin secretion by troglitazone and pioglitazone. The effects of PPAR-{alpha} agonists on insulin secretion remain unclear, but it is generally believed that these ligands do not influence insulin secretion when used clinically. However, in this present study, we have found that PPAR-{alpha} ligands (fenofibrate and fenofibric acid), a PPAR-{gamma} ligand (troglitazone), and an endogenous ligand of PPAR-{gamma} (15-deoxy-{Delta}12,14-PGJ2) inhibit the KATP channel directly and may induce insulin secretion. These results indicate the existence of a new pharmacological action of PPAR ligands when used clinically.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. Fenofibrate, fenofibric acid, and troglitazone were kindly provided by Kaken Pharmaceutical Co., Ltd. (Tokyo, Japan) and Sankyo Chemical Industries, Ltd. (Tokyo, Japan). The concentration of fenofibrate and fenofibric acid used in these experiments was based on the circulating concentration of fenofibric acid (Caldwell, 1989Go). The concentration of troglitazone used in this experiment was chosen because it has been shown to be clinically relevant in studies of peripheral insulin resistance (Shibata et al., 1993Go). Fetal bovine serum (FBS) was purchased from Invitrogen (Carlsbad, CA). F-12K medium in powder form was purchased from Flow Laboratories (Irvine, Scotland, UK). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture. HIT-T15 cells were purchased from Flow Laboratories. The cells were cultured in F-12K medium containing 7 mmol/l glucose, supplemented with 10% FBS, and incubated in a 95% O2/5% CO2 incubator at 37°C.

Electrophysiological Experiments. KATP currents were recorded in the whole-cell patch-clamp configuration using an Axo-patch-1D amplifier (Axon Instruments Inc., Union City, CA). The standard extracellular solution contained 160 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl2, 2 mmol/l CaCl2, 10 mmol/l HEPES, and 0.5 mmol/l glucose. The pH of the extracellular solution was adjusted to 7.4 with NaOH. The pipette solution contained 150 mmol/l KCl, 10 mmol/l HEPES, 11 mmol/l EGTA, 1 mmol/l MgCl2, and 1 mmol/l CaCl2. The pH of the pipette solution was adjusted to 7.2 with KOH. The resistance of patch pipettes when filled with the pipette solution ranged from 2 to 4 M{Omega}. Following gigaohm seal formation, negative pressure was applied to the pipette to rupture the membrane and establish the whole-cell mode. The KATP currents were measured by repeatedly applying ramp pulses from -90 to -50 mV. All the experiments were completed within 10 min after establishing the whole-cell configuration. One concentration of fenofibrate, fenofibric acid, troglitazone, or 15-deoxy-{Delta}12,14-PGJ2 was applied to each cell. The reversal potential of the HIT-T15 cell current was -60.8 mV under our experimental conditions. Electrophysiological experiments were performed at room temperature (22-25°C).

Binding Experiments. HIT-T15 cells were resuspended at a density of 1.5 x 107 cells/ml in glucose-free F-12K medium. Competitive inhibition assays were performed with 1.0 nmol/l [3H]glibenclamide, and various concentrations of fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 were incubated for 1 h at room temperature (Masuda et al., 1995Go). Incubation was terminated by rapid filtration under a vacuum with a cell harvester and Multi-Screen FC glass fiber filters (MultiScreen separation system; Milli-pore Corporation, Billerica, MA), and cells were washed three times with F-12K medium. The radioactivity of the filters was measured using a liquid scintillation counter (Top Count; PerkinElmer Life and Analytical Sciences, Boston, MA) after the addition of 10 ml of scintillation cocktail (Aquasol-2; PerkinElmer Life and Analytical Sciences). Binding inhibition was expressed as a percentage of [3H]glibenclamide-specific binding.

Insulin Secretion Study. For insulin secretion studies, cells were plated on 24-multiwell plates (1 x 105 cells per well). On the day of the experiment, the culture medium was completely aspirated and replaced with fresh medium containing fenofibrate, fenofibric acid, or 15-deoxy-{Delta}12,14-PGJ2.

The direct effect of fenofibrate (10-7-10-4 mol/l), fenofibric acid (10-7-10-4 mol/l), troglitazone (10-7-10-4 mol/l), and 15-deoxy-{Delta}12,14-PGJ2 (10-7-10-4 mol/l) on acute-phase insulin secretion was examined by the static incubation of HIT-T15 cells. Fenofibrate, fenofibric acid, and 15-deoxy-{Delta}12,14-PGJ2 were dissolved in dimethyl sulfoxide and methyl acetate with a final concentration of dimethyl sulfoxide or methyl acetate below 0.05% in the culture medium (F-12K medium without FBS), and the same concentration was used with all groups to avoid the influence of increased osmolarity on insulin secretion.

After reaching confluence, cells were washed twice with fresh F-12K medium and incubated for 10 min in 1 ml of experimental media. The medium was then aspirated, and the cells were centrifuged at 14000 rpm for 10 min. The supernatant was removed and frozen until radioimmunoassay of insulin concentration by commercial radioimmunoassay kits (Phadeceph Insulin; Pfizer Japan, Tokyo, Japan).

Data Analysis. The concentration dependence of fenofibrate and fenofibric acid on KATP channels was determined by fitting the following equation:

(1)

where I is the current at -50 mV in the presence of the ligands and Ic is the current at -50 mV measured in the control solution. Both I and Ic were obtained by subtracting the measured current amplitudes during an exposure to 100 µM tolbutamide. [X] is the concentration of fenofibrate, fenofibric acid, troglitazone, or 15-deoxy-{Delta}12,14-PGJ2; IC50 is the concentration at which inhibition is half-maximal; and h is the Hill coefficient (slope factor).

The relationships between drug concentrations and specific binding were fitted by the following equation:

(2)

where y is the specific binding; Ki is the concentration at which binding is half-maximal; [X] is the concentration of fenofibrate, fenofibric acid, troglitazone, or 15-deoxy-{Delta}12,14-PGJ2; h is the Hill coefficient; a is the upper plateau (maximum value for specific binding); and b is the lower plateau.

All data represent the mean ± S.E. The statistical analysis of the means was performed by analysis of variance, followed by Duncan's multiple range test for the individual comparisons of the means.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of PPAR-{alpha} Ligand (Fenofibrate) on KATP Channel Activity. Figure 1A (1) shows a HIT-T15 whole-cell current in response to voltage ramps from -90 to -50 mV in standard extracellular solution. As shown in the control current, the current amplitudes evoked by ramps were stable over 10 min under our experimental conditions. Subsequent application of tolbutamide (10-4 mol/l) reduced the conductance by 94.3 ± 5.2% (n = 36), suggesting that the currents principally reflect the activity of KATP channels. Application of an extracellular solution containing 22.2 mmol/l glucose did not affect KATP channel current, indicating that the intracellular complex including the glycolysis system is replaced by the pipette solution in the whole-cell mode. Therefore, factors such as glucose metabolism do not interfere with the KATP channel under our experimental condition.



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  		Fig. 1. Effect of fenofibrate on KATP channel current from HIT-T15 cells. KATP channel current was measured in response to voltage ramps from -90 to -50 mV using the standard whole-cell patch-clamp configuration. A, representative KATP channel current under our experimental conditions (1), effect of 10-4 mol/l fenofibrate on KATP channel current (2), and a section of control current trace in expanded time scale (3). The line above the current pulses represents the application time of fenofibrate. The dotted line indicates the zero current level. B, concentration-response curve for inhibition of KATP channel current by fenofibrate. The relationship between the fenofibrate concentration and macroscopic KATP current is expressed as a fraction of its amplitude in the absence of the drug (I/Ic). The currents fitted eq. 1: IC50 = 3.26 µmol/l, h = 1.14. Values represent means ± standard errors. *, p < 0.01 and +, p < 0.05 versus control; n = 8 in each group.

 

As shown in Fig. 1A (2), the KATP channel current activity was inhibited slowly after the application of fenofibrate (approximately 3-5 min after the application to reach maximal inhibition), and the inhibition continued after the washout of fenofibrate. We have also confirmed the depolarization of the {beta}-cell membrane potential after applying fenofibrate in current-clamp mode (data not shown). Figure 1C shows the relationship between fenofibrate concentrations and relative current of KATP channel currents. The inhibitory effect of the KATP channel current was maximum at the concentration of 1 x 10-4 mol/l fenofibrate. The IC50 value of inhibiting the KATP channel current by fenofibrate was 3.3 ± 0.5 µmol/l (n = 8), and the Hill coefficient was 1.1 ± 0.2.

Effect of PPAR-{alpha} Ligand (Fenofibric Acid) on KATP Channel Activity. Figure 2A (1) shows a HIT-T15 cell KATP channel current in response to voltage ramps from -90 to -50 mV in standard extracellular solution. Fenofibric acid, the active form of fenofibrate, irreversibly inhibited the KATP current, and maximal inhibition was reached 3 to 5 min after exposure [Fig. 2A (2)]. The concentration-dependent relationship of KATP channel current for fenofibric acid is shown in Fig. 2C. The IC50 value for fenofibric acid on KATP channels was 94 ± 0.5 µmol/l (n = 8), and the Hill coefficient was 2.1 ± 0.26.



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  		Fig. 2. Effect of fenofibric acid on KATP channel current from the HIT-T15 cells. KATP channel current was measured in response to the voltage ramps from -90 to -50 mV in the standard whole-cell configuration. A, example of the KATP channel current under our experimental conditions (1), effect of 10-4 mol/l fenofibric acid on KATP channel current (2), and a sample of control current trace in an expanded time scale (3). The line above the current pulses represents the application time of fenofibric acid. The dotted line indicates the zero current level. B, concentration-response curve for inhibition of KATP channel current by fenofibric acid. The relationship between the fenofibric acid concentration and macroscopic KATP current is expressed as a fraction of its amplitude in the absence of the drug (I/Ic). The currents fitted eq. 1: IC50 = 94 µmol/l, h = 2.1. Values represent means ± standard errors. *, p < 0.01 versus control; n = 8 in each group.

 

Effect of PPAR-{gamma} Ligand (Troglitazone) on KATP Channel Currents. Figure 3A (1) shows a control KATP channel current in response to voltage ramp pulses from -90 to -50 mV in standard extracellular solution. Troglitazone also irreversibly inhibited KATP channel current, and again maximal inhibition was obtained 3 to 5 min after exposure of the cells to the drug. [Fig. 3A (2)]. The concentration-effect relationship of troglitazone on the KATP currents is shown in Fig. 3C. The IC50 value for troglitazone was 2.1 ± 0.4 µmol/l (n = 8), and the Hill coefficient was 1.9 ± 0.32.



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  		Fig. 3. Effect of troglitazone on KATP channel current from the HIT-T15 cells. KATP channel current was measured in response to voltage ramps from -90 to -50 mV in the standard whole-cell configuration. A, example of the KATP channel current under our experimental conditions (1), effect of 10-4 mol/l troglitazone on KATP channel current (2), and a sample of control current trace in an expanded time scale (3). The line above the current pulses represents the application time of troglitazone. The dotted line indicates the zero current level. B, concentration-response curve for inhibition of KATP channel current by troglitazone. The relationship between the troglitazone concentration and macroscopic KATP current is expressed as a fraction of its amplitude in the absence of the drug (I/Ic). The currents fitted eq. 1: IC50 = 2.1 µmol/l, h = 1.9. Values represent means ± standard errors. *, p < 0.01 versus control; n = 8 in each group.

 

Effect of Endogenous Ligand of PPAR-{gamma} (15-Deoxy-{Delta}12,14-PGJ2) on KATP Channel Currents. Figure 4A (1) shows a representative KATP channel current recorded from an HIT-T15 cell in standard extracellular solution. 15-Deoxy-{Delta}12,14-PGJ2 had effects similar to the other PPAR agonists since the KATP current became irreversibly inhibited, and maximal inhibition was reached approximately 3 to 5 min after the exposure [Fig. 4A (2)]. The concentration-effect curve for 15-deoxy-{Delta}12,14-PGJ2 inhibition of KATP currents is shown in Fig. 4C. The IC50 value for 15-deoxy-{Delta}12,14-PGJ2 inhibition was 7.3 ± 0.96 µmol/l (n = 8), and the Hill coefficient was 1.0 ± 0.13.



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  		Fig. 4. Effect of 15-deoxy-{Delta}12,14-PGJ2 on KATP channel current from the HIT-T15 cells. KATP channel current was measured in response to voltage ramps from -90 to -50 mV in the standard whole-cell configuration. A, example of the KATP channel current under our experimental conditions (1), effect of 4 x 10-5 mol/l 15-deoxy-{Delta}12,14-PGJ2 on KATP channel current (2), and a sample of control current trace in an expanded time scale (3). The line above the current pulses represents the application time of fenofibric acid. The dotted line indicates the zero current level. B, concentration-response curve for inhibition of KATP channel current by 15-deoxy-{Delta}12,14-PGJ2. The relationship between the 15-deoxy-{Delta}12,14-PGJ2 concentration and macroscopic KATP current is expressed as a fraction of its amplitude in the absence of the drug (I/Ic). The currents fitted eq. 1: IC50 = 7.3 µM, h = 1.0. Values represent means ± standard errors. *, p < 0.01 versus control; n = 8 in each group.

 

Inhibition Binding Assay with PPAR-{alpha} Ligands. To examine the possibility that PPAR-{alpha} ligands may bind to the same site as glibenclamide, we performed an inhibition binding assay with 1.0 nmol/l [3H]glibenclamide and various concentrations of fenofibrate or fenofibric acid. The equilibrium dissociation constant (KD) of [3H]glibenclamide was 0.18 nmol/l in HIT-T15 cells, consistent with the value of 0.22 nmol/l reported by Schwanstecher et al. (1992Go).

As shown in Fig. 5A, fenofibrate inhibited the binding of [3H]glibenclamide at concentrations higher than unlabeled glibenclamide. Fenofibrate inhibited the binding of [3H]glibenclamide at concentrations higher than 1 x 10-7 mol/l (74.3 ± 3.2% at 1 x 10-6 mol/l, 63.5 ± 1.8% at 1 x 10-5 mol/l, and 56.3 ± 4.3% at 1 x 10-4 mol/l, n = 3), whereas fenofibric acid failed to inhibit the binding of [3H]glibenclamide at the concentrations tested (Fig. 5B). The results suggest that fenofibrate binds to the same site as glibenclamide at these concentrations, but fenofibric acid may occupy a separate site.



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  		Fig. 5. Inhibition of the specific [3H]glibenclamide binding to HIT-T15 cells by increasing concentrations of unlabeled fenofibrate (A) and fenofibric acid (B). Fenofibrate inhibited the binding of [3H]glibenclamide at higher concentrations than the unlabeled glibenclamide, whereas fenofibric acid had no effect. Values represent the means ± standard error. *, p < 0.01 and +, p < 0.05 versus control; n = 3 in each group.

 

Inhibition Binding Assay with PPAR-{gamma} Ligands. Similarly, PPAR-{gamma} ligands 15-deoxy-{Delta}12,14-PGJ2 and troglitazone inhibited [3H]glibenclamide binding at concentrations higher than unlabeled glibenclamide. Inhibition by 15-deoxy-{Delta}12,14-PGJ2 to HIT-T15 cells was observed at concentrations higher than 1 x 10-6 mol/l (60.2 ± 0.1% at 5 x 10-6 mol/l, 50.0 ± 0.1% at 1 x 10-5 mol/l, and 40.0 ± 0.01% at 5 x 10-5 mol/l, n = 3) (Fig. 6A). Troglitazone inhibited [3H]glibenclamide binding at concentrations higher than 1 x 10-6 mol/l (79.9 ± 3% at 1 x 10-5 mol/l and 52.7 ± 6.5% at 1 x 10-4 mol/l, n = 3) (Fig. 6B). These results suggest that both troglitazone and 15-deoxy-{Delta}12,14-PGJ2 bind to the same site as glibenclamide.



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  		Fig. 6. Inhibition of specific [3H]glibenclamide binding to HIT-T15 cells by increasing concentrations of unlabeled 15-deoxy-{Delta}12,14-PGJ2 (A) and troglitazone (B). 15-Deoxy-{Delta}12,14-PGJ2 and troglitazone inhibited the binding of [3H]glibenclamide at higher concentrations than the unlabeled glibenclamide. Values represent the means ± standard error. *, p < 0.01 and +, p < 0.05 versus control; n = 3 in each group.

 

Effect of PPAR-{alpha} Ligands (Fenofibrate and Fenofibric Acid) and PPAR-{gamma} Ligand (15-Deoxy-{Delta}12,14-PGJ2) on Acute-Phase Insulin Secretion. Figure 7 shows the acute-phase insulin secretion after static incubation with fenofibrate (10-7-10-4 mol/l) (Fig. 7A), fenofibric acid (10-7-10-4 mol/l) (Fig. 7B), troglitazone (10-7-10-4 mol/l) (Fig. 7C), and 15-deoxy-{Delta}12,14-PGJ2 (10-7-10-4 mol/l) (Fig. 7D) for 10 min in F-12K medium containing 7 mmol/l glucose.



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  		Fig. 7. Effects of 10-min HIT-T15 cell exposure to fenofibrate (A), fenofibric acid (B), troglitazone (C), and 15-deoxy-{Delta}12,14-PGJ2 (D) on acute-phase insulin secretion. Insulin secretion was stimulated at concentrations over 10-6 mol/l in fenofibrate, at a concentration of 10-4 mol/l in fenofibric acid, at a concentration of 10-4 mol/l in troglitazone, and at concentrations over 10-5 mol/l in 15-deoxy-{Delta}12,14-PGJ2. The glucose concentration in the medium used in this study was 7 mmol/l. Values represent means ± standard errors. *, p < 0.01 and +, p < 0.05 versus control; n = 8 in each group.

 

Acute-phase insulin secretion in the absence of fenofibrate was 2.3 ± 0.4 ng/ml (n = 8). Fenofibrate significantly stimulated insulin secretion at concentrations of 10-6 mol/l (3.6 ± 0.3 ng/ml, n = 8), 10-5 mol/l (4.0 ± 0.6 ng/ml, n = 8), and 10-4 mol/l (4.0 ± 0.3 ng/ml, n = 8) (Fig. 7A).

The effect of fenofibric acid, the active form of fenofibrate, on insulin secretion was also examined. Under drug-free conditions, insulin secretion was 2.0 ± 0.1 ng/ml (n = 8). Fenofibric acid only gave a statistically significant increase in insulin secretion at a concentration of 10-4 mol/l (2.6 ± 0.1 ng/ml, n = 8) (Fig. 7B).

These stimulatory effects of fenofibrate and fenofibric acid on insulin secretion were also observed at a glucose concentration of 3 mmol/l, where the amount of acute-phase insulin secretion in control medium was 1.2 ± 0.3 ng/ml (n = 8). Fenofibrate significantly increased the amount of insulin secreted at concentrations of 10-5 mol/l (2.1 ± 0.2 ng/ml, n = 8; p < 0.05 versus control) and 10-4 mol/l (3.3 ± 0.7 ng/ml, n = 8; p < 0.01 versus control). In the fenofibric acid experiment, secretion under control conditions was also low (1.0 ± 0.8 ng/ml, n = 8), but fenofibric acid only significantly stimulated the insulin secretion at a concentration of 10-4 mol/l (2.1 ± 0.7 ng/ml, n = 8; p < 0.05 versus control).

Exposures to the PPAR-{gamma} ligand troglitazone for 10 min also enhanced insulin secretion. Under drug-free conditions, insulin secretion was 1.5 ± 0.3 ng/ml (n = 8), whereas in the presence of 10-4 mol/l troglitazone, insulin secretion was 2.6 ± 0.1 ng/ml (n = 8; Fig. 7C).

The effects of the endogenous PPAR-{gamma} ligand 15-deoxy-{Delta}12,14-PGJ2 on insulin secretion was also tested. Under control conditions, insulin secretion was 2.9 ± 0.3 ng/ml; 10-5 mol/l 15-deoxy-{Delta}12,14-PGJ2 increased secretion to 4.9 ± 0.6 ng/ml, and 10-4 mol/l 15-deoxy-{Delta}12,14-PGJ2 increased secretion to 6.4 ± 0.5 ng/ml (Fig. 7D).


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The present studies demonstrated that both PPAR-{alpha} ligands (fenofibrate and fenofibric acid) and PPAR-{gamma} ligands (troglitazone and 15-deoxy-{Delta}12,14-PGJ2) inhibit KATP channel and induce acute-phase insulin secretion. Fenofibrate, 15-deoxy-{Delta}12,14-PGJ2, and troglitazone inhibited the binding of [3H]glibenclamide. These results indicated that PPAR-{alpha} and -{gamma} ligands have a direct effect on the {beta}-cell membrane, which may be independent of the PPAR pathway in the cytoplasm.

Glibenclamide and tolbutamide reportedly show different patterns in inhibiting KATP channel current. Glibenclamide is known to inhibit pancreatic {beta}-cell KATP channel current irreversibly (Sturgess et al., 1988Go; Gribble et al., 1998Go; Ashcroft and Gribble, 1999Go). In contrast, tolbutamide inhibits pancreatic {beta}-cell KATP channel current in a reversible manner (Sturgess et al., 1988Go; Gribble et al., 1998Go; Ashcroft and Gribble, 1999Go; Babenko et al., 1999Go). In the present study, we have shown that fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 all inhibit the KATP channel current, and the inhibition continues after the removal of the ligands. This indicates that fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 may inhibit the KATP channel in a manner similar to glibenclamide. In addition, the fact that fenofibrate, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 inhibit the binding of [3H]glibenclamide suggests that these PPAR ligands may interact directly with SUR. Therefore, we speculate that fenofibrate, troglitazone, and 15-deoxy-{Delta}12,14-PGJ2 stimulate insulin secretion by binding to SUR and inhibiting the KATP channel, similar to sulfonylureas. Although the binding site of these ligands on SUR may not necessarily be the same as that for glibenclamide, we propose that they may bind to a site on SUR1 or related molecules and consequently close the channel. However, the fact that these PPAR ligands inhibit the binding of [3H]glibenclamide raises the possibility that these PPAR ligands may weaken the effect of glibenclamide when administered together in clinical use.

In this study, we have shown that fenofibrate, but not fenofibric acid, inhibited the binding of [3H]glibenclamide. We speculate that the structural difference between fenofibrate and fenofibric acid may contribute to these different effects (Fig. 8). Fenofibrate is rapidly converted into the carboxic acid form fenofibric acid by esterases in the liver and plasma (Caldwell, 1989Go). A chemical moiety lacking in the structure of fenofibric acid compared with fenofibrate could be important in the binding inhibition of [3H]glibenclamide. The fact that fenofibric acid also has an inhibitory effect on KATP channels suggests that fenofibric acid has an inhibitory effect on KATP channels by a different mechanism from that of fenofibrate, 15-deoxy-{Delta}12,14-PGJ2, and troglitazone.



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  		Fig. 8. Structure of fenofibrate and fenofibric acid.

 

The finding that fenofibrate stimulated insulin secretion and inhibited KATP channel activity may not be consistent with the fact that fenofibrate does not perturb glucose homeostasis. To date, there are no reports of hypoglycemia from patients administrating fenofibrate as a treatment for hyperlipidemia. Fenofibrate is rapidly converted into fenofibric acid in the liver and plasma, and blood fenofibrate declines to undetectable levels (Caldwell, 1989Go). It has been assumed that the pharmacologically relevant form of fenofibrate is the acid form. In this study, we have shown that fenofibric acid stimulated insulin secretion only at a concentration of 10-4 mol/l and that the magnitude of insulin secretion stimulated by fenofibric acid was lower than fenofibrate. The IC50 value for inhibition of KATP channels by fenofibric acid was 9.4 x 10-5 mol/l, a value much higher than the actual circulating concentration of fenofibric acid (Caldwell, 1989Go). Therefore, fenofibrate may hardly influence actual insulin secretion in clinical use.

Holness et al. (2003Go) reported that a 24-h application of the PPAR-{alpha} agonist WY 14,643 (pirinixic acid) opposed insulin secretion elicited by high fat feedings in rats. Tordjman et al. (2002Go) also reported that overexpression of PPAR-{alpha} in INS-1 cells, an insulinoma cell line, decreased basal and glucose-stimulated insulin secretion. These two reports clearly demonstrated that chronically activating PPAR-{alpha} will suppress insulin secretion. We also observed that the insulin secretion stimulated by fenofibrate or fenofibric acid does not persist when these compounds were applied for more than 2 h in HIT-T15 cells (data not shown). These chronic effects on {beta}-cells of PPAR-{alpha} ligands may be produced by the mechanism of a PPAR-{alpha}-dependent pathway, and this pathway may not be involved in the acute effects that we demonstrated in this investigation.

It is well known that there are PPAR-independent effects exerted by PPAR-{gamma} ligands such as 15-deoxy-{Delta}12,14-PGJ2 and troglitazone. These include effects caused by microvascular endothelial cells and skeletal muscle (Brunmair et al., 2001Go; Jozkowicz et al., 2001Go). PPAR-{gamma}-independent effects of 15-deoxy-{Delta}12,14-PGJ2 and ciglitazone are involved in the generation of interleukin-8 by the human microvascular endothelial cell line (Jozkowicz et al., 2001Go). Troglitazone may have a PPAR-{gamma}-independent effect on skeletal muscle fuel metabolism (Brunmair et al., 2001Go). It has been shown that 15-deoxy-{Delta}12,14-PGJ2 has a direct effect on the I{kappa}B kinase complex and nuclear factor-{kappa}B activation in HeLa cells, which do not express PPAR-{gamma} (Rossi et al., 2000Go). It was also reported that 15-deoxy-{Delta}12,14-PGJ2 impairs cytokine signaling by inhibiting I{kappa}B and stimulating interferon-{gamma} and nitric oxide generation independently of PPAR-{gamma} in pancreatic {beta}-cells (Weber et al., 2004Go). These cytokines or generation of nitric oxide may also influence insulin secretion from pancreatic {beta}-cells. The effects of PPAR ligands in our study are clearly different from the PPAR-independent effects reported by Weber et al. (2004Go). Our study revealed that PPAR ligands stimulate insulin secretion and inhibit KATP channel activity in the whole-cell configuration in a very short period of time (10 min). Cytokine signaling or nitric oxide generation cannot stimulate insulin secretion within 10 min under our experimental conditions, because intracellular complexes including cytokines are replaced by a pipette solution during the whole-cell mode. In addition, Ogawa et al. (1999Go) reported that application of a low concentration of troglitazone (3 µM) does not induce insulin secretion in HIT-T15 cells. Our results showed that troglitazone inhibited KATP channel and stimulated acute-phase insulin secretion with a comparatively high concentration. Therefore, we speculate that troglitazone may inhibit KATP channel and induce insulin secretion only at the higher concentration. Our study strongly indicates the existence of the PPAR-independent pathway of its ligands, which directly inhibit the KATP channel in the {beta}-cell membrane. However, the concentration relation between PPAR ligands and KATP channel inhibition does not completely correlate with the concentration relation between PPAR ligands and the amount of acute-phase insulin secretion. Recently, there have been reports indicating that voltage-dependent K+ channels and ATP-dependent Ca2+ pumping, in addition to the KATP channel, control the {beta}-cell membrane potential and threshold for insulin secretion (Kanno et al., 2002Go; MacDonald and Wheeler, 2003Go). The lack of complete correlation in concentration between KATP channel inhibition and insulin secretion in our study may be explained by these factors other than KATP channel that control the membrane potential. Further studies are required to determine the precise mechanism of PPAR ligands on acute-phase insulin secretion.

The present finding that troglitazone inhibited KATP channels differs from the results reported by Masuda et al. (1995Go). They did not observe the inhibitory effect of troglitazone on KATP channel activity, despite the stimulation of insulin secretion. This may be because troglitazone was applied at a concentration lower (10-6 mol/l) than we used; thus, it would have been difficult to detect the inhibitory effect on the KATP channels.

Heron et al. (1998Go) recently reported that {alpha}-endosulfine may act as an endogenous regulator of {beta}-cell KATP channel activity and insulin secretion. {alpha}-Endosulfine is a peptide that was purified from bovine brain in the quest for an endogenous ligand for the SUR in brain (Virsolvy-Vergine et al., 1988Go). In the present study, we have shown that the endogenous ligand for PPAR-{gamma} 15-deoxy-{Delta}12,14-PGJ2 may bind to SUR and inhibit KATP channels. Prostaglandins (PGs) are known to play an important role in biological processes, and PGD2 is a major cyclooxygenase product in a variety of tissues having a marked biological effect (Giles and Leff, 1988Go). It has been reported that PGD2 undergoes dehydration to the J2 series in both in vitro and in vivo experiments (Fitzpatrick and Wynalda, 1983Go; Kikawa et al., 1984Go). One of the J2 series, 15-deoxy-{Delta}12,14-PGJ2, is known to be the endogenous ligand of PPAR-{gamma} (Kilwer et al., 1995). The present study adds the possibility that 15-deoxy-{Delta}12.14-PGJ2 may also act as an endogenous regulator of {beta}-cell KATP channel activity and insulin secretion by binding directly to SUR1.

KATP channels are known to exist not only in pancreatic {beta}-cells but also in various tissues such as neurons, cardiac muscle, skeletal muscle, and smooth muscle. In these tissues, different types of SURs serve as the regulatory subunits of KATP channels in association with pore-forming Kir6.x subunits: SUR1 in pancreatic {beta}-cells and neurons, SUR2A in cardiac and skeletal muscles, and SUR2B in smooth muscle (Inagaki et al., 1995Go, 1996Go, 1997Go; Sakura et al., 1995Go; Chutkow et al., 1996Go; Ishimoto et al., 1996Go; Yamada et al., 1997Go; Karschin et al., 1998Go). It is intriguing to consider whether PPAR ligands may have similar effects on [3H]glibenclamide binding to the SUR isoforms other than SUR1.


   Acknowledgements
 
We thank Kaken Pharmaceutical Co., Ltd. and Sankyo Chemical Industries for generously supplying fenofibrate, fenofibric acid, and troglitazone for this study. We also thank Dr. J. D. Lippiat (University of Oxford, Oxford, UK) for comments on this manuscript.


   Footnotes
 
doi:10.1124/jpet.104.067249.

ABBREVIATIONS: KATP, ATP-sensitive potassium; PPAR, peroxisome proliferator-activated receptor; 15-deoxy-{Delta}12,14-PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; FBS, fetal bovine serum; PG, prostaglandin.

Address correspondence to: Dr. Kenju Shimomura, Department of Medicine and Molecular Science, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511 Japan. E-mail: kenju.shimomura{at}physiol.ox.ac.uk


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
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