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Tacrolimus Impairment of Insulin Secretion in Isolated Rat Islets Occurs at Multiple Distal Sites in Stimulus-Secretion Coupling

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka City 812-8582, Japan

Address all correspondence and requests for reprints to: Dr. Masanori Iwase, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka City 812-8582, Japan. E-mail: iwase{at}intmed2.med.kyushu-u.ac.jp.

	Abstract

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Tacrolimus causes posttransplant diabetes mellitus, although the pathogenetic mechanisms remain controversial. We studied the mechanism of tacrolimus-induced impairment of insulin secretion using isolated rat pancreatic islets. Tacrolimus caused reductions in DNA and insulin contents per islet during 7-d culture. Tacrolimus time-dependently suppressed glucose-stimulated insulin secretion, and at a therapeutic concentration of 0.01 µmol/liter, it suppressed glucose-stimulated insulin secretion to 32 ± 5% of the control value after 7-d incubation. Tacrolimus did not change islet glucose utilization and oxidation, ATP production, insulin mRNA expression, or the capacity for high glucose to increase intracellular Ca²⁺, but altered the rapid frequency oscillations of Ca²⁺ concentration. Tacrolimus suppressed insulin secretion stimulated by mitochondrial fuel (combination of L-leucine and L-glutamine, and -ketoisocaproate) and glibenclamide, but not by L-arginine. Tacrolimus suppressed insulin secretion induced by carbachol and by a protein kinase C agonist in the presence or absence of extracellular Ca²⁺. Under stringent Ca²⁺-free conditions, tacrolimus did not affect mastoparan-induced insulin secretion, but suppressed its glucose augmentation. Our results suggest that tacrolimus impairs glucose-stimulated insulin secretion downstream of the rise in intracellular Ca²⁺ at insulin exocytosis, and that protein kinase C-mediated (Ca²⁺-dependent and independent) and Ca²⁺-independent GTP signaling pathways may be involved. However, tacrolimus-induced impaired insulin secretion was reversed 3 d after removal of the drug. Our study demonstrated that tacrolimus impairs insulin secretion at multiple steps in stimulus-secretion coupling.

	Introduction

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TACROLIMUS (FK506) is a hydrophobic macrocyclic lactone isolated from the fermentation broth of Streptomyces tsukubaensis from Mt. Tsukuba in Japan (1). Tacrolimus is a potent immunosuppressant and promotes graft survival in organ transplantation. The mechanism of T cell immunosuppression is through inhibition of Ca²⁺/calmodulin-dependent Ser/Thr phosphatase, calcineurin (protein phosphatase-2B), which controls the activation and translocation of the transcriptional cofactor, nuclear factor of activated T cells (NFAT), to the nuclei and persistence within the nuclei, and thus suppression of IL-2 transcription in T cells (2, 3). However, the incidence of posttransplant diabetes mellitus (PTDM) in renal transplant recipients treated with tacrolimus ranges from 10–30% in western countries (4) and is 31.4% in Japan (5). PTDM is associated with increased cardiovascular diseases and infection in transplant recipients. With better survival rates, PTDM has been recognized as a more serious complication than previously considered. In addition, tacrolimus has been increasingly used for the treatment of other diseases, such as dermatoses and inflammatory bowel disease (6, 7).

The exact mechanism(s) of the diabetogenic effect of tacrolimus has not yet been elucidated. To date, however, at least four possible mechanisms have been proposed (8, 9, 10, 11, 12, 13, 14, 15, 16). First, animal experiments and examination of human pancreas allograft biopsies indicate that long-term tacrolimus treatment causes cytoplasmic swelling, vacuolization, and apoptosis of ß-cells (8, 9). The ß-cell toxicity of tacrolimus seems to be related to blood drug levels, although the exact mechanism of such cytotoxicity has not yet been determined. Second, cyclic ADP-ribose (cADPR), a possible second messenger for glucose-stimulated insulin secretion, binds FK506-binding protein 12.6 (FKBP 12.6) in islet microsomes, leading to increased Ca²⁺ release via ryanodine receptor and enhanced insulin secretion (10, 11). Tacrolimus suppresses this pathway by binding with FKBP 12.6 (12). Third, it was reported that insulin gene transcription is regulated by NFAT, which is activated by Ca²⁺-dependent calcineurin in ß-cells (13). Tacrolimus suppressed glucose-stimulated insulin gene expression, leading to reduced insulin synthesis and contents. Lastly, insulin storage ß-granules are transported to the cell surface along microtubules, driven by a motor molecule such as kinesin (14, 15). In this regard, kinesin heavy chain is activated through dephosphorylation mediated by calcineurin. Suppression of calcineurin activity inhibits dephosphorylation of kinesin heavy chain as well as the second phase of glucose-stimulated insulin secretion (16).

The present study was designed to elucidate the mechanism(s) of tacrolimus-induced impairment of insulin secretion using isolated rat pancreatic islets. We studied islet cell viability, insulin secretion stimulated by various substances, insulin production and contents, islet glucose metabolism, and the intracellular free Ca²⁺ concentration ([Ca²⁺]i). The results suggest that tacrolimus impairs glucose-stimulated insulin secretion downstream of the rise in [Ca²⁺]i at insulin exocytosis and the possible involvement of protein kinase C (PKC)-mediated (Ca²⁺-dependent and independent) and Ca²⁺-independent GTP signaling pathways.

	Materials and Methods

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Islet isolation and culture
Male Sprague Dawley rats, weighing 250–350 g, were used for islet isolation (Kyudo Co., Kumamoto, Japan). Islets were isolated by collagenase digestion (17). The islets were hand-picked under a stereomicroscope (Leica MZ8, Leica, Heerbrugg, Switzerland) and transferred to RPMI 1640 medium (Sigma-Aldrich Corp., St. Louis, MO) containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich Corp.), and 11.0 mmol/liter glucose (Wako, Osaka, Japan) in 60 x 15-mm petri dishes (Sumilon, Akita, Japan). The islets (100/dish) were cultured in 4 ml RPMI 1640-based medium with different concentrations of tacrolimus (Fujisawa Pharmaceutical Co., Tokyo, Japan) in 0.1% ethanol or with the solvent alone as a control at 37 C in humidified air containing 5% CO₂. The culture medium was changed overnight after islet isolation and every other day thereafter. All experiments were performed according to the guidelines of the animal experimentation ethics committee of Kyushu University.

Assessment of islet cell viability
One hundred freshly isolated islets were cultured in the dish, and the number of total islets in the same batch was counted after 7-d culture with 0.1 or 1.0 µmol/liter tacrolimus. Subsequently, all islets in the culture dish were homogenized in water, a fraction of the homogenate was analyzed for DNA content (Gene Quant, Pharmacia, Cambridge, UK), and another fraction was mixed with acid ethanol for insulin extraction to determine insulin content. After incubation with 0.1 or 1.0 µmol/liter tacrolimus for 7 d, necrosis and early apoptosis of islet cells were determined using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (MBL, Nagoya, Japan). For a positive control, islets were cultured with 5 µg/ml cycloheximide (Nacalai Tesque, Kyoto, Japan) for 3 d. Islets 150–200 µm in diameter were transferred to 200 µl binding buffer, and 1 µl annexin V-FITC and 1 µl propidium iodide (PI) were added. After incubation at room temperature for 5 min in the dark, islets were observed under fluorescence stereomicroscope system (Leica MZ125, Leica, Wetzlar, Germany) equipped with a dual filter set for FITC and PI, and images were captured by computer-linked camera (CoolSNAP camera, Nippon Roper Co., Chiba, Japan) by the investigator who was blinded to the experimental groups. Cells in early apoptosis were stained green by annexin V-FITC, and necrotic cells were stained red by PI. Islet cell viability was assessed by calculating the percent area of apoptosis (green) and necrosis (red) per whole islet using the MacSCOPE system (version 2.5, Mitani Co., Chiba, Japan) in a blinded manner. In each experiment (n = 3), 10 islets were measured in control and tacrolimus-treated groups in a parallel fashion. However, this method could not measure the central part of the whole islet. Thus, caspase-3 activity was measured using the caspase-3/CPP32 colorimetric protease assay kit (MBL) in all islets in each culture dish. Briefly, islets were lysed and sonicated on ice. Then, 5 µl 4 mmol/liter DEVD-pNA substrate (Asp-Glu-Val-Asp linked to the colorichrome p-nitroanilide) were added to 50 µl blank and samples in 50 µl 2x reaction buffer containing 10 mmol/liter dithiothreitol. After incubation at 37 C for 2 h, absorbance at 405 nm was measured by a microplate reader (MPR-A 4I, Tosho, Tokyo, Japan). Enzyme activity was corrected for by DNA contents and expressed as a percentage of the control value. As a positive control, we used 5 µg/ml cycloheximide-treated islets.

Static insulin secretion
Measurements were carried out in Krebs-Ringer bicarbonate buffer (KRB) containing 118.4 mmol/liter NaCl, 4.69 mmol/liter KCl, 1.2 mmol/liter MgSO₄, 1.18 mmol/liter KH₂PO₄, 2.4 mmol/liter CaCl₂, and 20 mmol/liter NaHCO₃ (equilibrated with 95% O₂ and 5% CO₂, pH 7.4) supplemented with 0.2% BSA (fraction V). Islets 150–200 µm in diameter, randomly picked from the culture dish, were preincubated at 37 C for 30 min in KRB at 3.3 mmol/liter glucose. In glucose stimulation experiments, triplicate batches of five islets were incubated at 37 C in 0.5 ml KRB in the presence of 3.3 mmol/liter glucose for 1 h under continuous shaking and then in the presence of 16.7 mmol/liter glucose for another hour. Experiments were always performed in a parallel fashion in control and tacrolimus-treated islets. The reversibility of the effects of tacrolimus was studied 3 or 7 d after removal of the drug. In another experiment, triplicate batches of five islets were incubated at 37 C in 0.5 ml KRB for 1 h under continuous shaking, without or with one of the following test materials, 10 mmol/liter L-arginine (Sigma-Aldrich Corp.), 10 mmol/liter L-leucine (Wako Biochemicals, Osaka, Japan) combined with10 mmol/liter L-glutamine (Sigma-Aldrich Corp.), 10 mmol/liter -ketoisocaproate (KIC; Sigma-Aldrich Corp.), 1 µmol/liter thapsigargin (Sigma-Aldrich Corp.), 100 µmol/liter carbachol (Sigma-Aldrich Corp.), 5 µmol/liter forskolin (Wako Biochemicals), and 500 nmol/liter 12-O-tetradecanoylphorbol 13-acetate (TPA) (Wako Biochemicals), and 1 µmol/liter glibenclamide (Aventis Pharmaceuticals, Frankfurt, Germany). For Ca²⁺-free conditions, KRB buffer devoid of Ca²⁺ was used with 1 mmol/liter EGTA (Ca²⁺-free KRB/EGTA). After preincubation, triplicate batches of five islets were washed in Ca²⁺-free KRB/EGTA and then incubated at 37 C in 0.5 ml Ca²⁺-free KRB/EGTA for 1 h under continuous shaking without or with 10 µmol/liter mastoparan (Sigma-Aldrich Corp.) or 500 nmol/liter TPA. After incubation, the medium was removed and analyzed for insulin by RIA (Eiken RIA kit, Tokyo, Japan), and islet DNA and insulin contents were measured as described above. DNA and insulin contents in islets removed after the insulin secretion study did not differ in the batches of control and tacrolimus-treated cells in each experiment. Insulin secretion was expressed as microunits per islet per hour when comparing the results at the same culture day. However, when comparing results on different culture days, insulin secretion was expressed as the percentage of insulin contents.

Quantification of insulin mRNA by real-time PCR
Total RNA of all islets in the culture dish (150 islets) was extracted using Isogen (Wako Biochemicals) (18). Total RNA (0.5 µg) was reverse transcribed to cDNA by oligo(deoxythymidine)₁₅ primer (Promega Corp., Madison, WI) using Moloney murine leukemia virus reverse transcriptase (Ready-To-Go RT-PCR beads, Amersham Pharmacia Biotech, Piscataway, NJ). PCR primers were originally designed from the common gene sequences of rat insulin I and II (GenBank accession no. J00747) and ß-actin (GenBank accession no. J00691). To detect rat insulin mRNA, we amplified a 200-bp fragment using the oligonucleotide pair 5'-GTACCTGGTGTGTGGGGAAC-3' and 5'-CCAGTTGGTAGAGGGAGCA-3' as sense and antisense primers, respectively. For ß-actin mRNA, we amplified a 260-bp fragment, using the oligonucleotides 5'-CCTGTATGCCTCTGGTCGTA-3' and 5'-CCATCTCTTGCTCGAAGTCT-3'. PCR amplification was performed with the LightCycler-FastStart DNA Master SYBR Green I kit (Roche, Indianapolis, IN) in a PCR containing 0.5 µmol/liter of each primer, 2.5 mmol/liter MgCl₂ for insulin, 4 mmol/liter MgCl₂ for ß-actin, and 2 µl sample. Amplification and detection were carried out in a LightCycler instrument (LightCycler quick system 330, Roche Molecular Biochemicals, Mannheim, Germany). Amplification was performed for 35 cycles, with the following cycle parameters: denaturation (95 C for 15 sec), annealing (65 C for 8 sec for insulin and for 11 sec for ß-actin), and elongation (72 C for 10 sec). Rat insulin or ß-actin PCR products, which had been purified previously by GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech) and quantified, were used as standards to construct each standard curve with serial dilutions at 10-fold intervals. Quantification was carried out using LightCycler analysis software. The standard curve was constructed by plotting the log of copy number vs. the crossing points. ß-Actin amplification was used to rule out failure in each RNA purification, reverse transcriptase reaction, and PCR amplification reaction and to control variation in cDNA quantity among samples. All results were expressed as the ratio of the copy number of insulin to that of ß-actin.

Islet glucose utilization
Islet glucose utilization was measured by the method of Ashcroft et al. (19). After preincubation in KRB at 3.3 mmol/liter glucose for 30 min at 37 C, triplicate batches of 10 islets, 150–200 µm in diameter, were placed into a 1-ml glass cup with 16.7 mmol/liter glucose and 1 µCi D-[5-³H]glucose (Amersham Pharmacia Biotech) in 40 µl KRB, pH 7.4. Each cup with its content was placed in a 20-ml glass scintillation vial containing 500 µl distilled water, bubbled with 95% O₂ and 5% CO₂, and sealed airtight with a rubber stopper. The vials were shaken for 120 min at 37 C. Islet glucose metabolism was stopped with 100 µl 0.5 mol/liter HCl injected through the stopper into the cup. Parallel incubations were performed without islets. ³H₂O was collected for 18–24 h at 37 C. The cup was removed, and 5 ml scintillation fluid (Scintisol EX-H, Dojindo, Kumamoto, Japan) were added to the vial. After vortexing, radioactivity was counted in a liquid scintillation counter (LSC 1000, Aloka, Tokyo, Japan).

Islet glucose oxidation
After preincubation in KRB at 3.3 mmol/liter glucose for 30 min at 37 C, triplicate batches of 10 islets, 150–200 µm in diameter, were placed into a 1-ml glass cup with 16.7 mmol/liter glucose and 1 µCi D-[U-¹⁴C]glucose (Amersham Pharmacia Biotech) in 100 µl KRB, pH 7.4 (19). Each cup with its content was placed in a 20-ml glass scintillation vial, bubbled with 95% O₂ and 5% CO₂, and then sealed airtight with a rubber stopper. The vials were shaken for 90 min at 37 C. Islet glucose metabolism was stopped with 100 µl 0.05 mmol/liter antimycin A (Wako Biochemicals) dissolved in 70% (vol/vol) ethanol, which was injected through the stopper into the cup. In the next step, 250 µl hyamine hydroxide (Packard, Meriden, CT) were injected into the vial. CO₂ was released from the incubation medium with 100 µl 0.4 mol/liter Na₂HPO₄ (Wako Biochemicals) solution (pH 6.0) injected into the cup. To allow the CO₂ to be trapped by hyamine hydroxide, the vials were shaken for another 120 min at 37 C. Parallel incubations were performed without islets. The cup was removed, and 5 ml scintillation fluid were added to the vial. After vortexing, radioactivity was counted in a liquid scintillation counter.

Measurement of islet ATP content
ATP content was determined by a luminometric method (20). Islets were preincubated in KRB at 3.3 mmol/liter glucose at 37 C for 30 min, then triplicate batches of 10 islets, 150–200 µm in diameter, were placed in 0.5 ml KRB at 3.3 or 16.7 mmol/liter glucose under continuous shaking for 60 min at 37 C. The reaction was stopped by the addition of 0.5 ml trichloroacetic acid to a final concentration of 5%. The tubes were immediately mixed with vortex and then sonicated in ice-cold water for 3 min. They were centrifuged (2000 x g for 3 min), and a fraction (0.7 ml) of the supernatant was mixed with 1 ml water-saturated diethyl ether. The ether phase containing trichloroacetic acid was discarded. This step was repeated four times. After the extracts (0.1 ml) were diluted with 0.1 ml 20 mmol/liter HEPES, pH 7.4, with NaOH, they were frozen at –80 C until assays. The thawed extracts (50 µl) were added to 150 µl of a solution of 20 mmol/liter HEPES (pH 7.75) and 3 mmol/liter MgCl₂. The ATP concentration in the solutions was measured by adding 100 µl luciferin-luciferase solution (Enliten ATP Assay System, Promega) to a fraction sample (20 µl) in a bioluminometer (MiniLumat LB 9506, Berthold, Bad Wildbad, Germany). To construct a standard curve, blanks and ATP standards were run through the entire procedure, including the extraction steps.

Measurement of islet [Ca²⁺]i
Measurements (n = 5) were carried out in Krebs-Ringer bicarbonate buffer containing 128.8 mmol/liter NaCl, 4.8 mmol/liter KCl, 1.2 mmol/liter MgSO₄, 1.2 mmol/liter KH₂PO₄, 2.5 mmol/liter CaCl₂, 5 mmol/liter NaHCO₃, and 10 mmol/liter HEPES (KRBH; equilibrated with NaOH, pH 7.4) supplemented with 0.1% BSA. Islets were attached to a glass-bottom culture dish (MatTek Co., Ashland, MA) that had been precoated with 100 µg/ml poly-L-lysine (Sigma-Aldrich Corp.) in RPMI 1640-based medium containing 11 mmol/liter glucose for 1 h at 37 C in 95% O₂-5% CO₂ incubator. The islets were then washed twice in KRBH containing 2.8 mmol/liter glucose, loaded with 3 µmol/liter Fura 2/AM (Dojindo) (21) in KRBH containing 2.8 mmol/liter glucose, and then incubated at 37 C for 30 min. The islets were placed on the stage of an inverted microscope (Leica DM IRB) and superfused at 2 ml/min at 37 C with KRBH with 2.8 mmol/liter glucose, then with 16.7 mmol/liter glucose. The Fura-2-loaded single whole islet was illuminated by excitation at 340 nm (F340) and 380 nm (F380) alternately every 6–8 sec through a x10 fluorite objective, and the emission signals at 510 nm were detected by a CCD camera (C4742-95-12ER, Hamamatsu Photonics, Hamamatsu, Japan). The F340/F380 ratio image was produced using a dual excitation microfluorescence system (Aquacosmos version 1.3, Hamamatsu Photonics). The peripheral portion of the islet was omitted for the measurement, and ß-cells were confirmed by applying 100 µmol/liter tolbutamide (Aventis Pharmaceuticals). Two to four islets of similar size (110 µm in diameter) were used, and the total number of islets measured was 14 in the control group and 11 in the tacrolimus group. In each experiment, measurements were made in control and tacrolimus-treated islets in a parallel fashion, and F340/F380 ratio was used as described for [Ca²⁺]i. For analysis of [Ca²⁺]i oscillations, the trend of [Ca²⁺]i baseline was corrected by taking the difference between the two continuous data, and then the amplitude and period of [Ca²⁺]i oscillations were calculated.

Statistical analysis
Values are expressed as the mean ± SEM. Statistical significance was evaluated by unpaired or paired t test, or ANOVA with Scheffé’s F test as a post hoc test. P < 0.05 was considered significant.

	Results

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Effects of tacrolimus on islet recovery, islet DNA, insulin contents, and mRNA expression
As shown in Table 1, tacrolimus at 0.1 or 1.0 µmol/liter did not affect islet recovery after 7-d culture. However, islet DNA contents were significantly reduced in tacrolimus-treated islets compared with the control, when all islets in the culture dish were used. Islet insulin contents were also reduced in tacrolimus-treated islets. However, there were no significant differences in DNA and insulin contents between 0.1 and 1.0 µmol/liter tacrolimus. Tacrolimus did not significantly alter insulin contents relative to DNA contents or insulin mRNA relative to ß-actin mRNA.

Effects of tacrolimus on glucose-stimulated insulin secretion
Tacrolimus did not alter insulin secretion at 3.3 mmol/liter glucose after 3- and 7-d culture (Fig. 1). However, it significantly suppressed insulin secretion in response to 16.7 mmol/liter glucose when used at concentration of 0.1 µmol/liter for 3 d (percentage of control, 60 ± 5%) and further suppressed it when used at 0.1 and 1.0 µmol/liter for 7 d (percentage of control, 39 ± 4% and 37 ± 4%, respectively). The latter experiments were performed at 7 d of culture. Figure 2 shows the effects of various concentrations of tacrolimus on glucose-stimulated insulin. Tacrolimus suppressed insulin secretion at 0.001 µmol/liter (percentage of control, 56 ± 5%; P = NS vs. control) and at 0.01 µmol/liter (percentage of control, 32 ± 5%; P < 0.05 vs. control), but no further suppression was noted at higher concentrations. Thus, 0.1 µmol/liter tacrolimus was used in the following experiments unless otherwise stated. At 7 d of culture with various concentrations of tacrolimus, islet DNA and insulin contents were not different in control and tacrolimus-treated islets of similar size used in insulin secretion studies (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Time-dependent effects of tacrolimus on 3.3 mmol/liter () and 16.7 mmol/liter () glucose-stimulated insulin secretion. Values are the mean ± SEM (n = 5). *, P < 0.05; **, P < 0.01 (vs. control).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effects of various concentrations of tacrolimus on 3.3 mmol/liter () and 16.7 mmol/liter () glucose-stimulated insulin secretion. Values are the mean ± SEM (n = 3–4). *, P < 0.05 vs. control.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Effects of tacrolimus on insulin secretion stimulated by 10 mmol/liter L-arginine at various glucose concentrations. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 5). *, P < 0.05 vs. control.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Effects of tacrolimus on insulin secretion stimulated by the combination of 10 mmol/liter L-leucine and 10 mmol/liter L-glutamine, and 10 mmol/liter KIC in the absence of glucose. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 4). *, P < 0.05, **, P < 0.01 (vs. control).

View larger version (10K): [in this window] [in a new window]   FIG. 5. Effects of tacrolimus on insulin secretion stimulated by 1 µmol/liter glibenclamide at 3.3 mmol/liter glucose. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 4). *, P < 0.05 vs. control.

View larger version (11K): [in this window] [in a new window]   FIG. 6. Effects of tacrolimus on insulin secretion stimulated by 1 µmol/liter thapsigargin (Tg) and/or 100 µmol/liter carbachol (CCh) at 5.5 mmol/liter glucose. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 5). **, P < 0.01 vs. control.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Effects of tacrolimus on insulin secretion induced by 5 µmol/liter forskolin at various glucose concentrations. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 3–4). *, P < 0.05, **, P < 0.01 (vs. control).

View larger version (12K): [in this window] [in a new window]   FIG. 8. Effects of tacrolimus on insulin secretion induced by 500 nmol/liter TPA in the presence or absence of extracellular Ca2+ at 3.3 mmol/liter glucose. , Control; , islets treated with 0.1 µmol/liter tacrolimus for 7 d. Values are the mean ± SEM (n = 4). *, P < 0.05, **, P < 0.01 (vs. control).

View larger version (14K): [in this window] [in a new window]   FIG. 9. Effects of tacrolimus on mastoparan-induced insulin secretion under stringent Ca2+-free conditions at 3.3 mmol/liter () and 16.7 mmol/liter () glucose. Values are the mean ± SEM (n = 4). *, P < 0.05 vs. control.

View larger version (14K): [in this window] [in a new window]   FIG. 10. Representative tracings of [Ca2+]i stimulated by 16.7 mmol/liter glucose in control islets (A) and islets treated with 0.1 µmol/liter tacrolimus for 7 d (B).

View larger version (15K): [in this window] [in a new window]   FIG. 11. Reversal of insulin secretion 3 and 7 d after the removal of tacrolimus (n = 4 each) in islets previously treated with 0.1 µmol/liter tacrolimus for 7 d (n = 14). Values are the mean ± SEM. **, P < 0.01 vs. control; ##, P < 0.01 vs. previously tacrolimus-treated islets.

	Discussion

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In the present study tacrolimus reduced DNA and insulin contents per islet, possibly by loss of ß-cells during 7-d culture. This is in line with histological changes observed in the pancreas in tacrolimus-treated rats (8) and biopsy specimens of transplanted pancreas in patients treated with tacrolimus (9). However, cell death (apoptosis and necrosis) or caspase-3 activity, one of the more distal proteases in the apoptosis pathway, was not increased in islet cells cultured with tacrolimus for 7 d. This disagreement may be due to a very slow process of ß-cell loss during 7-d culture or to our insensitive methods to detect cell death. Calcineurin was reported to regulate apoptosis by acting as an antiapoptotic agent through dephosphorylation of Bcl-2 protein (22) or as a proapoptotic agent through dephosphorylation of Bcl-2/Bcl-X_L-associated death promoter (23). Our study suggests that long-term treatment with tacrolimus may impair ß-cell survival as well as insulin secretion by the remaining ß-cells, both of which contribute to the diabetogenicity of tacrolimus.

Tacrolimus suppressed glucose-stimulated insulin secretion without affecting islet glucose metabolism or the increase in [Ca²⁺]i. Insulin secretion induced by nonglucose mitochondrial fuel, such as the combination of leucine and glutamine, and KIC was also reduced by tacrolimus. In addition, glibenclamide-induced insulin secretion was suppressed, although an increase in islet ATP contents at 16.7 mmol/liter glucose was not affected by tacrolimus. These findings suggest that tacrolimus may suppress insulin secretion at the level of exocytosis. Recently, Donelan et al. (16) reported that calcineurin causes Ca²⁺-dependent dephosphorylation of kinesin heavy chain in ß-granules, and that the phosphorylation state of kinesin heavy chain was inversely proportional to glucose-stimulated insulin secretion. Kinesin attaches ß-granules to microtubulin and drives granules along the microtubules toward the plasma membrane. Glucose-induced insulin secretion was suppressed in mouse ß-cells treated with antisense oligonucleotide of kinesin heavy chain (15) and in isolated rat islets incubated with a relatively specific calcineurin inhibitor, cypermethrin, and adenovirus mediated the expression of a specific calcineurin inhibitor, CAIN (16). On the other hand, knockout mice of Rab3A, a small G protein, exhibited impaired glucose-stimulated insulin release, but preserved arginine-induced insulin secretion with unaltered islet glucose metabolism and [Ca²⁺]i (24). These features resemble the results obtained in our tacrolimus-treated islets. Calmodulin is a major Rab3A target effector protein, and Rab3A-calmodulin interaction on ß-granules provides a platform for the activation of calcineurin in response to increased [Ca²⁺]i. In Rab3A^–/– mice, calmodulin may not be sequestered to calcineurin to be activated, and consequently, kinesin may not be dephosphorylated and activated, leading to failure in replenishing the readily releasable pool of insulin secretory granules. Therefore, tacrolimus may prevent kinesin-mediated ß-granule transport and may lower the ß-granule docked at the plasma membrane downstream of the rise in [Ca²⁺]i.

Glucose stimulates phosphatidylinositol turnover and activates PKC (25), and the role of PKC as an amplifier of the glucose-generated signals to release insulin has been well established. Moreover, glibenclamide may stimulate insulin secretion partly by PKC activation (26). In the present study tacrolimus significantly suppressed insulin secretion stimulated by TPA-sensitive PKC in a Ca²⁺-dependent as well as a Ca²⁺-independent manner. In addition, tacrolimus suppressed insulin secretion induced by carbachol under nonstimulatory conditions of 5.5 mmol/liter glucose, which may be mediated by a PKC-dependent pathway other than the mobilization of Ca²⁺ from inositol 1,4,5-triphosphate-sensitive stores (27). It is conceivable that insulin secretion is determined by a balance between protein phosphorylation and dephosphorylation (28). In this context, it is interesting that tacrolimus, a protein phosphatase inhibitor, may induce PKC signaling deficiency, leading to impaired insulin secretion. The mechanism by which tacrolimus suppressed Ca²⁺-sensitive and Ca²⁺-insensitive PKC isoenzymes remains to be elucidated.

Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin release under stringent Ca²⁺-free conditions, possibly by activation of GTP-binding protein (29). Mastoparan has a unique mode of action, in that neither the activation of PKA and PKC nor the involvement of phospholipase A2 is required (30), and stimulates insulin secretion via activation of the Rho subfamily of small G proteins, such as Cdc42 (31) and Rac (32). Metz et al. (33) reported that the abnormality in mastoparan-sensitive G protein might mediate impaired insulin secretion in a model of genetically lean type 2 diabetes mellitus, GK rats. In the present study tacrolimus did not affect mastoparan-induced insulin secretion per se, but suppressed its glucose augmentation in a Ca²⁺-independent manner. Glucose augments posttranslational modifications (e.g. carboxyl methylation and geranyl geranylation) of specific G proteins (e.g. Cdc42, Rap1, and Rac) in a GTP-sensitive manner (34, 35), and glucose augmentation of mastoparan-stimulated insulin secretion is extremely dependent upon GTP (36). Therefore, tacrolimus may affect the activation of a specific G protein, which could contribute to the alteration in insulin secretion.

[Ca²⁺]i oscillations occur synchronously throughout the whole islet through intercommunication via gap junctions. There are fast (duration, 20 sec) and slow (duration, 4–7 min) [Ca²⁺]i oscillations in mouse islets, whereas the presence of [Ca²⁺]i oscillations is controversial in rat islets. Although irregular and slow [Ca²⁺]i oscillations (37) or fast oscillations (38) have been observed in rat islets, their origin and significance are not clear at present. In our experiment, the amplitude of fast oscillations was significantly smaller in tacrolimus-treated islets than in the controls. Altered glucose-stimulated oscillations in islet [Ca²⁺]i were also observed in islets isolated from Rab3A^–/– mice, showing the longer period of oscillation. Although the mechanism and significance of altered [Ca²⁺]i oscillations remain to be elucidated, these may be related to dysfunction of exocytosis in ß-cells.

cADPR produced from NAD by ADP ribosylcyclase is an important messenger for Ca²⁺-induced Ca²⁺ release and glucose-stimulated insulin secretion (10, 39). Tacrolimus inhibited the cADPR-mediated dissociation of FKBP 12.6 from ryanodine receptors and thus suppressed Ca²⁺ release from the endoplasmic reticulum, resulting in reduced insulin secretion. However, our study showed that tacrolimus failed to modulate islet the increase in [Ca²⁺]i stimulated by a high glucose concentration. One possibility for this disagreement may be that the inhibition of cADPR-FKBP 12.6 interaction in ryanodine receptors of islet microsomes required a micromoles per liter concentration of tacrolimus, with maximal reduction at 5 µmol/liter. In addition, calcineurin has been reported to regulate insulin gene transcription by activating NFAT, and tacrolimus at 5–10 µmol/liter completely prevented high glucose-induced activation of insulin gene promoter activity in the INS-1 cell line. In our study, however, islet insulin mRNA expression in tacrolimus-treated islets was not different from the control value at 7 d of culture. It was also reported that incubation of human islets with 0.001 µmol/liter tacrolimus for 5 d did not result in a decrease in insulin mRNA expression (40). The reason for this discrepancy is not clear at present, although the concentration of tacrolimus used, the cell types, and/or the experimental conditions may explain these differences.

Our study showed that 0.01 µmol/liter tacrolimus suppressed to 32% of the control insulin secretion stimulated by 16.7 mmol/liter glucose, and that higher concentrations of tacrolimus caused no further suppression. The dose-dependence study using rat islets cultured with various concentrations of tacrolimus for 24 h showed that 0.06 µmol/liter tacrolimus had the maximal inhibitory effect on glucose-stimulated insulin secretion (41). In marked contrast, Fuhrer et al. (42) demonstrated that insulin secretion was stimulated in an insulinoma cell line incubated with 0.05 or 0.08 µmol/liter tacrolimus for 30 min, although lower or higher concentrations of tacrolimus had no effect. This acute insulin release was blocked by diazoxide (an ATP-sensitive potassium channel opener), verapamil (a voltage-dependent Ca²⁺ channel blocker), and stringent Ca²⁺-free conditions. Tacrolimus acutely induced insulin secretion only in a narrow range of concentrations in an ATP-sensitive potassium channel-dependent manner. The apparent difference in acute and chronic effects of tacrolimus seems to be due to difference in the proportion of ß-cells affected by the drug.

In conclusion, chronic treatment with therapeutic concentrations of tacrolimus caused islet cell loss and impaired insulin secretion in isolated rat islets. The impaired insulin secretion might be at the level of exocytosis beyond the increase in [Ca²⁺]i and may be related to Ca²⁺-dependent calcineurin inhibition. In addition, the present study suggests the possible involvement of PKC-mediated (Ca²⁺-dependent and independent) and Ca²⁺-independent GTP signaling pathways. Thus, it seems that tacrolimus impairs ß-cell function at multiple steps in stimulus-secretion coup-ling. However, the defective insulin secretion was reversible after withdrawal of the drug, in agreement with clinical experience (4).

	Acknowledgments

 
We are grateful to K. Sekioka and J. Ishimatsu for technical assistance.

	Footnotes

 
This work was supported by in part by Grant-in-Aid for Science Research C11671128, 1999–2002, from the Ministry of Education, Science, Sports, and Culture of Japan.

Abbreviations: [Ca²⁺]i, Intracellular Ca²⁺; cADPR, cyclic ADP-ribose; FITC, fluorescein isothiocyanate; FKBP 12.6, FK506-binding protein 12.6; KIC, -ketoisocaproate; KRB, Krebs-Ringer bicarbonate buffer; NFAT, nuclear factor of activated T cell; PI, propidium iodide; PKA, protein kinase A; PKC, protein kinase C; PTDM, posttransplant diabetes mellitus; TPA, 12-O-tetradecanoylphorbol 13-acetate.

Received September 3, 2003.

Accepted for publication February 4, 2004.

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